WO1994001567A1 - Process for immobilizing enzymes to the cell wall of a microbial cell by producing a fusion protein - Google Patents

Abstract

A method is provided for immobilizing an enzyme, comprising immobilizing the enzyme or a functional part thereof to the cell wall of a microbial cell using recombinant DNA techniques. The enzyme is immobilized by linking it to the C-terminal part of a protein that ensures anchoring in the cell wall. Also provided is a recombinant polynucleotide comprising a structural gene encoding an enzyme protein, a part of a gene encoding the C-terminal part of a protein capable of anchoring in a eukaryotic or prokariotic cell wall, as well as a signal sequence, in addition to a chimeric protein encoded by the recombinant polynucleotide and a vector and a microorganism containing the polynucleotide. The microorganism is suitable for carrying out enzymatic processes on an industrial scale.

Description

PROCESS FOR IMMOBILIZING ENZYMES TO THE CELL WALL OF A MICROBIAL CELL BY PRODUCING A FUSION PROTEIN.

The present invention is in the field of conversion processes using immobilized enzymes, produced by genetic engineering.

Background of the invention

In the detergent, personal care and food products industry there is a strong trend towards natural ingredients of these products and to environmentally acceptable production processes. Enzymic conversions are very important for fulfilling these consumer demands, as these processes can be completely natural. Moreover enzymic processes are very specific and consequently will produce minimum amounts of waste products. Such processes can be carried out in water at mild temperatures and atmos¬ pheric pressure. However enzymic processes based on free enzymes are either quite expensive due to the loss of enzymes or require expensive equipment, like ultra- membrane systems to entrap the enzyme.

Alternatively enzymes can be immobilized either physically or chemically. The latter method has often the disadvantage that coupling is carried out using non-natural chemicals and in processes that are not attractive from an environmental point of view. Moreover chemical modification of enzymes is nearly always not very specific, which means that coupling can affect the activity of the enzyme negatively. Physical immobilization can comply with consumer demands, however also physical immobilization may affect the activity of the enzyme in a negative way. Moreover, a physically immobilized enzyme is in equilibrium with free enzyme, which means that in continuous reactors, according to the laws of thermodynamics, substantial losses of enzyme are unavoidable.

There are a few publications on immobilization of enzymes to microbial cells (see reference 1). The present invention provides a method for immobilizing enzymes to cell walls of microbial cells in a very precise way. Additionally, the immobilization does not require any chemical or physical coupling step and is very efficient. Some extracellular proteins are known to have special functions which they can perform only if they remain bound to the cell wall of the host cell. Often this type of protein has a long C-terminal part that anchors it in the cell wall. These C-terminal parts have very special amino acid sequences. A typical example is anchoring via C- terminal sequences enriched in proline (see reference 2). Another mechanism to anchor proteins in cell walls is that the protein has a glycosyl-phosphatidyl-inositol (GPI) anchor (see reference 3) and that the C-terminal part of the protein contains a substantial number of potential serine and threonine glycosylation sites. O-Glycosylation of these sites gives a rod-like conformation to the C-terminal part of these proteins. Another feature of these manno-proteins is that they seem to be linked to the glucan in the cell wall of lower eukaryotes, as they cannot be extracted from the cell wall with SDS, but can be liberated by glucanase treatment.

Summary of the invention

The present invention provides a method for immobilizing an enzyme, which comprises the use of recombinant DNA techniques for producing an enzyme or a functional part thereof linked to the cell wall of a host cell, preferably a microbial cell, and whereby the enzyme or functional fragment thereof is localized at the exterior of the cell wall. Preferably the enzyme or the functional part thereof is immobilized by linking to the C-terminal part of a protein that ensures anchoring in the cell wall. In one embodiment of the invention a recombinant polynucleotide is provided comprising a structural gene encoding a protein providing catalytic activity and at least a part of a gene encoding a protein capable of anchoring in a eukaryotic or prokaryotic cell wall, said part encoding at least the C-terminal part of said anchoring protein. Preferably the polynucleotide further comprises a sequence encoding a signal peptide ensuring secretion of the expression product of the polynucleotide. Such signal peptide can be derived from a glycosyl-phosphatidyl-inositol (GPI) anchoring protein, a -factor, α-agglutinin, invertase or inulinase, α-amylase of Bacillus, or a proteinase of lactic acid bacteria. The DNA sequence encoding a protein capable of anchoring in the cell wall can encode α-agglutinin, AGA1, FLO1 or the Major Cell Wall Protein of lower eukaryotes, or a proteinase of lactic acid bacteria. The recombinant polynucleotide is operably linked to a promoter, preferably an inducible promoter. The DNA sequence encoding a protein providing catalytic activity can encode a hydrolytic enzyme, e.g. a lipase, or an oxidoreductase, e.g. an oxidase. Another embodiment of the invention relates to a recombinant vector comprising a polynucleotide as described above. If such vector contains a DNA sequence encoding a protein providing catalytic activity, which protein exhibits said catalytic activity when present in a multimeric form, said vector can further comprise a second polynucleotide comprising a structural gene encoding the same protein providing catalytic activity combined with a sequence encoding a signal peptide ensuring secretion of the expression product of said second polynucleotide, said second polynucleotide being operably linked to a regulatable promoter, preferably an inducible or repressible promoter.

A further embodiment of the invention relates to a chimeric protein encoded by a polynucleotide as described above. Still another embodiment is a host cell, preferably a microorganism, containing a polynucleotide as described above or a vector as described above. If the protein providing catalytic activity exhibits said catalytic activity when present in a multimeric form, said host cell or microorganism can further comprise a second polynucleotide comprising a structural gene encoding the same protein providing catalytic activity combined with a sequence encoding a signal peptide ensuring secretion of the expression product of said second polynucleotide, said second polynucleotide being operably linked to a regulatable promoter, preferably an inducible or repressible promoter, and said second polynucleotide being present either in another vector or in the chromosome of said microorganism. Preferably the host cell or microorganism has at least one of said polynucleotides integrated in its chromosome. As a result of culturing such host cell or microorganism the invention provides a host cell, preferably a microorganism, having a protein as described above immobilized on its cell wall. The host cell or microorganism can be a lower eukaryote, in particular a yeast.

The invention also provides a process for carrying out an enzymatic process by using an immobilized catalytically active protein, wherein a substrate for said catalytically active protein is contacted with a host cell or microorganism according to the invention. Brief Description of the Figures

Figure 1: DNA sequence of the 6057 bp Htndlll fragment containing the complete

AGαl gene of S. cerevisiae (see SEQ ID NO: 1 and 2). The position of the unique zel site and the H dIII site used for the described constructions is specified in the header.

Figure 2: Schematic presentation of the construction of pUR2969. The restriction sites for endonucleases used are shown. Abbreviations used: AG-alpha-1: Gene expressing α-agglutinin from 5. cerevisiae amp: β-lactamase resistance gene PGKp: phosphoglyceratekinase promoter

PGKt: terminator of the same gene.

Figure 3: α-Galactosidase activity of 5. cerevisiae MT302/1C cells and culture fluid transformed with pSY13 during batch culture:

A: U/l α-galactosidase per time; the OD₅₃₀ is also shown B: α-galactosidase activity of free and bond enzyme expressed in U/OD₅₃₀.

Figure 4: α-Galactosidase activity of 5. cerevisiae MT302/1C cells and culture fluid transformed with pUR2969 during batch culture:

A: U/l α-galactosidase per time; the OD₅₃₀ is also shown

B: α-galactosidase activity of free and bond enzyme expressed in U/OD₅₃₀. Figure 5: Western analysis with anti α-galactosidase serum of extracellular fractions of cells of exponential phase (OD₅₃₀=2). The analyzed fractions are equivalent to 4 mg cell walls, (fresh weight):

A: MT302/1C expressing α-galactosidase, lane 1, growth medium lane 2, SDS extract of isolated cell walls lane 3, glucanase extract of SDS extracted cell walls;

B: MT302/1C expressing α-Gal-AGαl fusion protein, lane 1, growth medium lane 2, SDS extract of isolated cell walls lane 3, glucanase extract of SDS-extracted cell walls lane 4: Endo-Η treated glucanase extract. Figure 6: Immunofluorescent labelling (anti α-galactosidase) of MT302/1C cells in the exponential phase (OD₅₃₀=2) expressing the α -Gal- α-agglutinin fusion protein. Phase micrograph of intact cells A: overview B: detail.

Figure 7: Schematic presentation of the construction of pUR2970A, pUR2971A, pUR2972A, and pUR2973. The restriction sites for endonucleases used are indicated in the figure. PCR oligonucleotide sequences are mentioned in the text. Abbreviations used: AGal cds: coding sequence of α-agglutinin a-AGG=AGal: Gene expressing α-agglutinin from S. cerevisiae amp: β-lactamase resistance gene Pgal7=GAL7: GAL7 promoter lipolase: lipase gene of Hwnicola invSS: SUC2 signal sequence a-MF: prepro-α -mating factor sequence a-gal: α-galactosidase gene

LEU2d : truncated promoter of LEU2 gene; LETJ2 : LEU2 gene with complete promoter sequence. Figure 8: DNA sequence of a fragment containing the complete coding sequence of lipase B of Geotrichum candidum strain 335426 (see SEQ ID NO: 11 and 12). The sequence of the mature lipase B starts at nucleotide 97 of the given sequence. The coding sequence starts at nucleotide 40 (ATG).

Figure 9: Schematic presentation of the construction of pUR2975 and pUR2976. The restriction sites for endonucleases used are shown. Abbreviations used: a-AGG: Gene expressing α-agglutinin from 5. cerevisiae amp: β-lactamase resistance gene Pgal7=GAL7: GAL7 promoter invSS: SUC2 signal sequence a-MF: prepro-α-mating factor sequence

LEU2d: truncated promoter LEU2 gene lipolase: lipase gene of Humicola lipaseB: lipaseB gene of Geotrichum candidum. Figure 10: Schematic presentation of the construction of pUR2981 and pUR2982. The restriction sites for endonucleases used are shown. Abbreviations used: a-AGG = AG-alpha 1: Gene expressing α-agglutinin from S. cerevisiae mucor lipase: lipase gene of Rhizomucor miehei 2u: 2μm sequence

Pgal7 = GAL7: GAL 7 promoter invSS: SUC2 signal sequence a-MF: prepro-α-mating factor sequence lipolase: lipase gene of Humicola amp: β-lactamase resistance gene; LEU2d: truncated promoter LEU2 gene

LEU2 : LEU2 gene with complete promoter sequence. Figure 11: DNA sequence (2685 bases) of the 894 amino acids coding part of the

FLOl gene (see SEQ ID NO: 21 and 22), the given sequence starts with the codon for the first amino acid and ends with the stop codon.

Figure 12: Schematic presentation of plasmid pUR2990. Some restriction sites for en- donucleases relevant for the given cloning procedure are shown.

Figure 13: Schematic presentation of plasmid pUR7034.

Figure 14: Schematic presentation of plasmid pUR2972B.

Figure 15: Immunofluorescent labelling (anti-lipolase) of SU10 cells in the exponential phase (OD₅₃₀ = 0.5) expressing the lipolase/-α-agglutinin fusion protein. A: phase micrograph B: matching fluorescent micrograph

Detailed description of the invention

The present invention provides a method for immobilizing an enzyme, comprising immobilizing the enzyme or a functional part thereof to the cell wall of a host cell, preferably a microbial cell, using recombinant DNA techniques. In particular, the C- terminal part of a protein that ensures anchoring in the cell wall is linked to an enzyme or the functional part of an enzyme, in such a way that the enzyme is localized on or just above the cell surface. In this way immobilized enzymes are obtained on the surface of cells. The linkage is performed at gene level and is characterized in that the structural gene coding for the enzyme is coupled to at least part of a gene encoding an anchor-protein in such a way that in the expression product the enzyme is coupled at its C-terminal end to the C-terminal part of an anchor-protein. The chimeric enzyme is preferably preceded by a signal sequence that ensures efficient secretion of the chimeric protein. Thus the invention relates to a recombinant polynucleotide comprising a structural gene encoding a protein providing catalytic activity and at least a part of a gene encoding a protein capable of anchoring in a eukaryotic or prokaryotic cell wall, said part encoding at least the C-terminal part of said anchoring protein. The length of the C-terminal part of the anchoring protein may vary. Although the entire structural protein could be used, it is preferred that only a part is used, leading to a more efficient exposure of the enzyme protein in the medium surrounding the cell. The anchoring part of the anchoring protein should preferably be entirely present. As an example, about the C-terminal half of the anchoring protein could be used. Preferably, the polynucleotide further comprises a sequence encoding a signal peptide ensuring secretion of the expression product of the polynucleotide. The signal peptide can be derived from a GPI anchoring protein, α-factor, α-agglutinin, invertase or inulinase, α-amylase of Bacillus, or a proteinase of lactic acid bacteria. The protein capable of anchoring in the cell wall is preferably selected form the group of α-agglutinin, AGA1, FLOl (flocculation protein) or the Major Cell Wall Protein of lower eukaryotes, or a proteinase of lactic acid bacteria. The polynucleotide of the invention is preferably operably linked to a promoter, preferably a regulatable promoter, especially an inducible promoter.

The invention also relates to a recombinant vector containing the polynucleotide as described above, and to a host cell containing this polynucleotide, or this vector. In a particular case, wherein the protein providing catalytic activity exhibits said catalytic activity when present in a multimeric form, such as may be the case with oxidoreductases, dimerisation or multimerisation of the monomers might be a prerequisite for activity. The vector and/or the host cell can then further comprise a second polynucleotide comprising a structural gene encoding the same protein pro¬ viding catalytic activity combined with a sequence encoding a signal peptide ensuring secretion of the expression product of said second polynucleotide, said second polynucleotide being operably linked to a regulatable promoter, preferably an inducible or repressible promoter. Expression and secretion of the second polynucleotide after expression and secretion of the first polynucleotide will then result in the formation of an active multimer on the exterior of the cell wall. The host cell or microorganism preferably contains the polynucleotide described above, or at least one of said polynucleotides in the case of a combination, integrated in its chromosome.

The present invention relates in particular to lower eukaryotes like yeasts that have very stable cell walls and have proteins that are known to be anchored in the cell wall, e.g. α-agglutinin or the product of gene FLOl. Suitable yeasts belong to the genera Candida, Debatyomyces, Hansenula, KLuyveromyces, Pichia and Saccharomyces. Also fungi, especially Aspergillus, Penicillium and Wiizopus can be used. For certain applications also prokaryotes are applicable.

For yeasts the present invention deals in particular with genes encoding chimeric enzymes consisting of: a. the signal sequence e.g. derived from the α-factor-, the invertase-, the α- agglutinin- or the inulinase genes; b. structural genes encoding hydrolytic enzymes such as α-galactosidase, proteases, peptidases, pectinases, pectylesterase, rhamnogalacturonase, esterases and Upases, or non-hydrolytic enzymes such as oxidases; and c. the C-terminus of typically cell wall bound proteins such as α-agglutinin (see reference 4), AGA1 (see reference 5) and FLOl (see the non-prior published reference 6). The expression of these genes can be under the control of a constitutive promoter, but more preferred are regulatable, i.e. repressible or inducible promoters such as the GAL7 promoter for Saccharomyces, or the inulinase promoter for IQuyveromyces or the methanol-oxidase promoter for Hansenula.

Preferably the constructs are made in such a way that the new genetic information is integrated in a stable way in the chromosome of the host cell.

The invention further relates to a host cell, in particular a microorganism, having the chimeric protein described above immobilized on its cell wall. It further concerns the use of such microorganisms for carrying out an enzymatic process by contacting a substrate for the enzyme with the microorganism. Such a process may be carried out e.g. in a packed column, wherein the microorganisms may be supported on solid par¬ ticles, or in a stirred reactor. The reaction may be aqueous or non-aqueous. Where necessary, additives necessary for the performance of the enzyme, e.g. a co-factor, may be introduced in the reaction medium.

After repeated usage of the naturally immobilized enzyme system in processes, the performance of the system may decrease. This is caused either by physical denaturation or by chemical poisoning or detachment of the enzyme. A particular feature of the present invention is that after usage the system can be recovered from the reaction medium by simple centrifugation or membrane filtration techniques and that the thus collected cells can be transferred to a recovery medium in which the cells revive quickly and concomitantly produce the chimeric protein, thus ensuring that the surface of the cells will be covered by fully active immobilized enzyme. This regeneration process is simple and cheap and therefore will improve the economics of enzymic processes and may result in a much wider application of processes based on immobilized enzyme systems.

However, by no means the present invention is restricted to the reusability of the immobilized enzymes.

The invention will be illustrated by the following examples without the scope of the invention being limited thereto.

EXAMPLE 1 Immobilized α-galactosidase/α-agglutinin on the surface of S. cerevisiae.

The gene encoding α-agglutinin has been described by Lipke et al. (see reference 4). The sequence of a 6057 bp H dIII insert in pTZ18R, containing the whole AGαl gene is given in Figure 1. The coding sequence expands over 650 amino acids, including a putative signal sequence starting at nucleotide 3653 with ATG. The unique Nhel site cuts the DNA at position 988 of the given coding sequence within the coding part of amino acid 330, thereby separating the α-agglutinin into an N- terminal and a C-terminal part of about same size. Through digestion of pUR2968 (see Figure 2) with Nhel/Hindlll a 1.4 kb fragment was released, containing the sequence information of the putative cell wall anchor. For the fusion to α-galactosidase the plasmid pSY16 was used, an episomal vector based on YEplac 181, harbouring the α-galactosidase sequence preceded by the SUC2 invertase signal sequence and placed between the constitutive PGK promoter and PGK terminator. The Styl site, present in the last nine base-pairs of the open reading frame of the α-galactosidase gene, was ligated to the Nhel site of the AGal gene fragment. To ensure the in frame fusion, the Styl site was filled in and the 5' overhang of the Nhel site was removed, prior to ligation into the Styl/ Hindlll digested pSY13 (see Figure 2). To verify the correct assembly of the new plasmid, the shuttle vector was transformed into E. coli JM109 (recAl supE44 endAl hsdR17 gyrA96 relAl thi {lac-proAB) F [traD36 proAB^* lacP lacZ--M15]) (see reference 7) by the transformation protocol described by Chung et al. (see reference 8). One of the positive clones, designated pUR2969, was further characterized, the DNA isolated and purified according to the Quiagen protocol and subsequently characterized by DNA sequencing. DNA sequencing was mainly performed as described by Sanger et al. (see reference 9), and Hsiao (see reference 10), here with the Sequenase version 2.0 kit from United States Biochemical Company, according to the protocol with T7 DNA polymerase (Amersham International pic) and [^SJdATPαS (Amersham International pic: 370 MBq/ml; 22 TBq/mmol). This plasmid was then transformed into S. cerevisiae strain MT302/1C according to the protocol from Klebe et al. (see reference 11).

Yeast transformants were selected on selective plates, lacking leucine, on with 40 μl (20mg/ml DMF). X-α-Gal (5-bromo-4-chloro-3-indolyl-α-D-glucose, Boehringer Mannheim) was spread, to directly test for α-galactosidase activity (see reference 12). To demonstrate the expression, secretion, localization and activity of the chimeric protein the following analyses were performed: 1. Expression and secretion

S. cerevisiae strain MT302/1C was transformed with either plasmid pSY13 containing the α-galactosidase gene of Cyamopsis tetragonoloba or plasmid pUR2969 containing the α-galactosidase/α-agglutinin fusion construct. During batch culture α-galactosidas activities were determined for washed cells and growth medium. The results are given in Figure 3 and Figure 4. The α-galactosidase expressed from yeast cells containing plasmid pSY13 was almost exclusively present in the growth medium (Figure 3A), whereas the α-galactosidase- α-agglutinin fusion protein was almost exclusively cell associated (Figure 4A). Moreover, the immobilized, cell wall-associated, α-galacto- sidase-α-agglutinin fusion enzyme had retained the complete activity over the whole incubation time, while the secreted and released enzyme lost about 90% of the activity after an incubation of 65 hours. This indicates, that the immobilization of the described enzyme into the cell wall of yeast protects the enzyme against inactivation, presumably through proteinases, and thereby increases the stability significantly. Further insight into the localization of the different gene products was obtained by Western analysis. Therefore, cells were harvested by centrifugation and washed in 10 mM Tris.HCl, pH 7.8; 1 mM PMSF at 0°C and all subsequent steps were performed at the same temperature. Three ml isolation buffer and 10 g of glass beads were added per gram of cells (wet weight). The mixture was shaken in a Griffin shaker at 50% of its maximum speed for 30 minutes. The supernatant was isolated and the glass beads were washed with 1 M NaCl and 1 mM PMSF until the washes were clear. The supernatant and the washes were pooled. The cell walls were recovered by centrifugation and were subsequently washed in 1 mM PMSF. Non-covalently bound proteins or proteins bound through disulphide bridges were released from cell walls by boiling for 5 minutes in 50 mM Tris.HCl, pH 7.8; containing 2 % SDS, 100 mM EDTA and 40 mM β-mercaptoethanol. The SDS- extracted cell walls were washed several times in 1 mM PMSF to remove SDS. Ten mg of cell walls (wet weight) were taken up in 20 1 100 mM sodium acetate, pH 5.0, containing 1 mM PMSF. To this, 0.5 mU of the β-l,3-glucanase (Laminarase; Sigma L5144) was used as a source of β-l,3-glucanase) was added followed by incubation for 2 hours at 37 °C. Subsequently another 0.5 mU of β-l,3-glucanase was added, followed by incubation for another 2 hours at 37 °C.

Proteins were denatured by boiling for 5 minutes preceding Endo-H treatment. Two mg of protein were incubated in 1 ml 50 mM potassium phosphate, pH 5.5, containing 100 mM β-mercaptoethanol and 0.5 mM PMSF with 40 mU Endo-H (Boehringer) for 48 hours at 37 °C. Subsequently 20 mU Endo-H were added followed by 24 hours of incubation at 37 °C.

Proteins were separated by SDS-PAGE according to Laemmli (see reference 13) in 2.2.-20% gradient gels. The gels were blotted by electrophoretic transfer onto Immobilon polyvinylidene-difluoride membrane (Millipore) as described by Towbin et al. (see reference 14). In case of highly glycosylated proteins a subsequently mild periodate treatment was performed in 50 mM periodic acid, 100 mM sodium acetate, pH 4.5, for several hours at 4 °C. All subsequent incubations were carried out at room temperature. The blot was blocked in PBS, containing 0.5% gelatine and 0.5% Tween-20, for one hour followed by incubation for 1 hour in probe buffer (PBS, 0.2% gelatine, 0.1% Tween-20) containing 1:200 diluted serum. The blot was subsequently washed several times in washing buffer (PBS; 0.2% gelatine; 0.5% Tween-20) followed by incubation for 1 hour in probe-buffer containing ¹²⁵I-labelled protein A (Amersham). After several washes in washing buffer, the blot was air-dried, wrapped in Saran (Dow) and exposed to X-omat S film (Kodak) with intensifying screen at -70 °C. An Omnimedia 6cx scanner and the Adobe Photoshop programme were used to quantify the amount of labelled protein. The results of the various protein isolation procedures from both transformants are given in Figure 5. While for the transformants comprising the pSY13 plasmid the overall mass of the enzyme was localized in the medium, with only minor amounts of enzyme more entrapped than bond in the cell wall (Figure 5A) -which could completely be removed by SDS extrac¬ tion- the fusion protein was tightly bound to the cell wall; with only small amounts of α-galactosidase/α-agglutinin delivered into the surrounding culture fluid or being SDS extractable. In contrast to the laminarinase extraction of cell walls from cells expressing the free α-galactosidase, where no further liberation of any more enzyme was observed, identical treatment of fusion enzyme expressing cells released the overall bulk of the enzyme. This indicates that the fusion protein is intimately associated with the cell wall glucan in S. cerevisiae, like α-agglutinin, while α-galactosi¬ dase alone is not. The subsequently performed EndoH treatment showed a heavy glycosylation of the fusion protein, a result, entirely in agreement with the described extended glycosylation of the C-terminal part of α-agglutinin. 2. Localization Immunofluorescent labelling with anti-α-galactosidase serum was performed on intact cells to determine the presence and distribution of α-galactosidase/α-agglutinin fusion protein in the cell wall. Immunofluorescent labelling was carried out without fixing according to Watzele et al. (see reference 15). Cells of OD₅₃₀=2 were isolated and washed in TBS (10 mM Tris.HCl, pH 7.8, containing 140 mM NaCl, 5 mM EDTA and 20 μg/ml cycloheximide). The cells were incubated in TBS + anti-α-galactosidase serum for 1 hour, followed by several washings in TBS. A subsequent incubation was carried out with FITC-conjugated anti-rabbit IgG (Sigma) for 30 minutes. After washing in TBS, cells were taken up in 10 mM Tris.HCl, pH 9.0, containing 1 mg/ml p-phenylenediamine and 0.1 % azide and were photographed on a Zeiss 68000 microscope. The results of these analysis are given in Figure 6, showing clearly that the chimeric α-galactosidase/α-agglutinin is localized at the surface of the yeast cell. Buds of various sizes, even very small ones very uniformly labelled, demonstrates that the fusion enzyme is continuously incorporated into the cell wall throughout the cell cycle and that it instantly becomes tightly linked. 3. Activity

To quantitatively assay α-galactosidase activity, 200 μl samples containing 0.1 M sodium-acetate, pH 4.5 and 10 mM p-nitrophenyl-α-D-galactopyranoside (Sigma) were incubated at 37 °C for exactly 5 minutes. The reaction was stopped by addition of 1 ml 2% sodium carbonate. From intact cells and cell walls, removed by centrifu¬ gation and isolated and washed as described, the α-galactosidase activity was calcu¬ lated using the extinction coefficient of p-nitrophenol of 18.4 cm²/mole at 410 nm. One unit was defined as the hydrolysis of 1 μmole substrate per minute at 37 °C.

Table 1. Distribution of free and immobilized α-galactosidase activity in yeast cells

α-Galactosidase activity (U/g F.W. cells^) Expressed Growth Intact Isolated protein medium cells cell walls α-galactosidase 14.7 0.37 0.01 αGal/αAGG fusion protein 0.54 13.3 10.9

Transformed MT302/1C cells were in exponential phase (OD₅₃₀=2). One unit is defined as the hydrolysis of 1 μmole of p-nitrophenyl-α-D-galactopyranoside per minute at 37 °C.

The results are summarized in Table 1. While the overall majority of α-galactosidase was distributed in the culture fluid, most of the fusion product was associated with the cells, primarily with the cell wall. Taking together the results shown in Figures 3 to 6 and in Table 1, it could be calculated that the enzymatic α-galactosidase activity of the chimeric enzyme is as good as that of the free enzyme. Moreover, during stationary phase, the activity of the α-galactosidase in the growth medium decreased, whereas the activity of the cell wall associated α-galactosidase α-agglutinin fusion remained constant, indicating that the cell associated fusion protein is protected from inactivation or proteolytic degradation.

N.B. The essence of this EXAMPLE was published during the priority year by M.P. Schreuder et al. (see reference 25).

EXAMPLE 2A Immobilized Humicola lipase/α-agglutinin on the surface of S. cerevisiae. (inducible expression of immobilized enzyme system)

The construction and isolation of the 1.4 kb Nhel/Hindlll fragment containing the C- terminal part of α-agglutinin has been described in EXAMPLE 1. Plasmid pUR7021 contains an 894 bp long synthetically produced DNA fragment encoding the lipase of Humicola (see reference 16 and SEQ ID NO: 7 and 8), cloned into the EcoRI/H dlll restriction sites of the commercially available vector pTZ18R (see Figure 7). For the proper one-step modification of both the 5' end and the 3' end of the DNA part coding for the mature lipase, the PCR technique can be applied.

Therefore the DNA oligonucleotides lipol (see SEQ ID NO: 3) and lipo2 (see SEQ ID NO: 6) can be used as primers in a standard PCR protocol, generating an 826 bp long DNA fragment with an Eagl and a Hindlll restriction site at the ends, which can be combined with the larger part of the Eagl/Hindlϊl digested pUR2650, a plasmid containing the α-galactosidase gene preceded by the invertase signal sequence as des¬ cribed earlier in this specification, thereby generating plasmid pUR2970A (see Figure 7).

PCR oligonucleotides for the in-frame linkage of Humicola lipase and the C- terminus of α agglutinin.

a: PCR oligonucleotides for the transition between SUC2 signal sequence and the N-terrninus of lipase.

>mature lipase Eagl E V S Q D L F primer lipol: 5'-GGG GCG GCC GAG GTC TCG CAA GAT CTG GA-3' lipase: 3'-TAA GCA GCT CITICI CIAIGI AIGICI GITITI CITIGI GIAICI CITIG TTT-5*

(non-coding strand, see SEQ ID NO: 4)

b: PCR oligonucleotides for the in frame transition between C-terminus of lipase and C-terminal part of α-agglutinin.

F G L I G T C L lipase: 5*-TTC GGG TTA ATT GGG ACA TGT CTT TAG TGC GA-3' (co. strand) I primer 3'-C|C|C| A|A|T| T|A|A| C|C|C| H TGT AnCAi GIAIAI CGA TCG GAA TTC GAACCCC-5' lipo2: Nhel Hindlll

(for the part of the lipase coding strand see SEQ ID NO: 5)

Through the PCR method a iel site will be created at the end of the coding sequence of the lipase, allowing the in-frame linkage between the DNA coding for lipase and the DNA coding for the C-terminal part of α-agglutinin. Plasmid pUR2970A can then be digested with Miel and H dIII and the 1.4 kb Nhel/Hindlll fragment containing the C-terminal part of α-agglutinin from plasmid pUR2968 can be combined with the larger part of Nhel and Hindlll treated plasmid pUR2970A, resulting in plasmid pUR2971A. From this plasmid the 2.2 kb Eαgl/Hindl l fragment can be isolated and ligated into the Eαgl- and Ht/idlll-treated pUR2741, whereby plasmid pUR2741 is a derivative of pUR2740 (see reference 17), where the second Eαgl restriction site in the already inactive Tet resistance gene was deleted through Nrul/Sall digestion. The Sail site was filled in prior to religation. The ligation then results in pUR2972A containing the GAL7 promoter, the invertase signal sequence, the chimeric lipase/α-agglutinin gene, the 2 μm sequence, the defective Leu2 promo¬ ter and the Leu2 gene. This plasmid can be used for transforming S. cerevisiae and the transformed cells can be cultivated in YP medium containing galactose as an inducer without repressing amounts of glucose being present, which causes the expression of the chimeric lipase/α-agglutinin gene. The expression, secretion, localization and activity of the chimeric lipase/α-agglutinin can be analyzed using similar procedures as given in EXAMPLE .1.

In a similar way variants of Humicola lipase, obtained via rDNA techniques, can be linked to the C-terminal part of α-agglutinin, which variants can have a higher stability during (inter)esterification processes.

EXAMPLE 2B Immobilized Humicola lipase/α-agglutinin on the surface of S. cerevisiae (inducible expression of immobilized enzyme system) EXAMPLE 2A describes a protocol for preparing a particular construct. Before carrying out the work it was considered more convenient to use the expression vector described in EXAMPLE 1, so that the construction route given in this EXAMPLE 2B differs on minor points from the construction route given in EXAMPLE 2A and the resulting plasmids are not identical to those described in EXAMPLE 2A. However, the essential gene construct comprising the promoter, signal sequence, and the structural gene encoding the fusion protein are the same in EXAMPLES 2A and 2B.

1. Construction

The construction and isolation of the 1.4 kb Nhel/Hindlll fragment encoding the C- terminal part of α-agglutinin cell wall protein has been described in EXAMPLE 1. The plasmid pUR7033 (resembling pUR7021 of EXAMPLE 2A) was made by treating the commercially available vector pTZ18R with EcoRI and H dlll and ligating the resulting vector fragment with an 894 bp long synthetically produced DNA EcoRI/H dIII fragment encoding the lipase of Humicola (see SΕQ ID NO: 7 and 8, and reference 16).

For the fusion of the lipase to the C-terminal, cell wall anchor-comprising domain of α-agglutinin, plasmid pUR7033 was digested with Eagl and H dIII, and the lipase coding sequence was isolated and ligated into the Eagl- and H dIII-digested yeast expression vector pSYl (see reference 27), thereby generating pUR7034 (see Figure 13). This is a 2μm episomal expression vector, containing the α-galactosidase gene described in EXAMPLE 1, preceded by the invertase (SUC2) signal sequence under the control of the inducible GALl promoter. Parallel to this digestion, pUR7033 was also digested with EcoRV and Htndlll, thereby releasing a 57 bp long DNA fragment, possessing codons for the last 15 car- boxyterminal amino acids. This fragment was exchanged against a small DNA frag¬ ment, generated through the hybridisation of the two chemically synthesized deoxyoligonucleotides SEQ ID NO: 9 and SEQ ID NO: 10. After annealing of both DNA strands, these two oligonucleotides essentially reconstruct the rest of the 3' coding sequence of the initial lipase gene, but additionally introduce downstream of the lipase gene a new Nhel restriction site, followed by a Htndlll site in close vicinity, whereby the first three nucleotides of the Nhel site form the codon for the last amino acid of the lipase. The resulting plasmid was designated pUR2970B. Subsequently, this construction intermediate was digested with Eagl and Nhel, the lipase encoding fragment was isolated, and, together with the 1.4 kb Nhel/HindUl fragment of pUR2968 ligated into the Eagl- and Hmdlll-cut pSYl vector. The outcome of this 3- point-ligation was called pUR2972B (see Figure 14), the final lipolase-α-agglutinin yeast expression vector.

This plasmid was used for transforming S. cerevisiae strain SU10 as described in reference 17 and the transformed cells were cultivated in YP medium containing galactose as the inducer without repressing amounts of glucose being present, which causes the expression of the chimeric lipase/α-agglutinin gene. 2. Activity

To quantify the lipase activity, two activity measurements with two separate substrates were performed. In both cases, SU10 yeast cells transformed with either plasmid pUR7034 or pSYl served as control. Therefore, yeast cell transformants containing either plasmid pSYl or plasmid pUR7034 or plasmid pUR2972B were grown up for 24h in YNB-glucose medium supplied with histidine and uracil, then diluted 1:10 in YP-medium supplied with 5% galactose, and again cultured. After 24h incubation at 30°C, a first measurement for both assays was performed.

The first assay applied was the pΗ stat method. Within this assay, one unit of lipase activity is defined as the amount of enzyme capable of liberating one micromole of fatty acid per minute from a triglyceride substrate under standard assay conditions (30 ml assay solution containing 38 mM olive oil, considered as pure trioleate, emulsified with 1:1 w/w gum arabic, 20 mM calcium chloride, 40 mM sodium chloride, 5 mM Tris, pH 9.0, 30°C) in a radiometer pH stat apparatus (pHM 84 pH meter, ABU 80 autoburette, TTA 60 titration assembly). The fatty acids formed were titrated with 0.05 N NaOH and the activity measured was based on alkali consumption in the interval between 1 and 2 minutes after addition of putative enzyme batch. To test for immobilized lipase activity, 1 ml of each culture was centrifuged, the supernatant was saved, the pellet was resuspended and washed in 1 ml 1 M sorbitol, subsequently again centrifuged and resuspended in 200 μl 1 M sorbitol. From each type of yeast cell the first supernatant and the washed cells were tested for lipase activity.

A: Lipase activity after 24h (LU/ml) cell bound culture fluid pSYl 5.9 8.8 pUR7034 24.1 632.0 pUR2972B-(l) 18.7 59.6 pUR2972B-(2) 24.6 40.5

The rest of the yeast cultures was further incubated, and essentially the same separation procedure was done after 48 hours. Dependent on the initial activity measured, the actual volume of the sample measured deviated between 25 μl and

150μl.

This series of measurements indicates, that yeast cells comprising the plasmid coding for the lipase- α-agglutinin fusion protein in fact express some lipase activity which is associated with the yeast cell. An additional second assay was performed to further confirm the immobilization of activity of lipase on the yeast cell surface. Briefly, within this assay, the kinetics of the PNP (=paranitrophenyl) release from PNP-butyrate is determined by measurement of the OD at 400 run. Therefore, 10 ml cultures containing yeast cells with either pSYl, pUR7034 or pUR2972B were centrifuged, the pellet was resuspended in 4 ml of buffer A (0.1 M NaOAc, pH 5.0 and 1 mM PMSF ), from this 4 ml 500μl was centrifuged again and resuspended in 500 μl PNB-buffer (20 mM Tris-HCl, pH 9.0, 20 mM CaC12, 25 mM NaCl), centrifuged once again, and finally resuspended in 400μl PNB buffer. This fraction was used to determine the cell bound fraction of lipase.

The remaining 3500μl were spun down, the pellet was resuspended in 4 ml A, to each of this, 40 μl laminarinase (ex mollusc, 1.25 mU/μl) was added and first incubated for 3 hours at 37°C, followed by an overnight incubation at 20°C. Then the reaction mixture, still containing intact cells, were centrifuged again and the supernatant was used to determined the amount of originally cell wall bound material released through laminarinase incubation. The final pellet was resuspended in 400μl PNP buffer, to calculate the still cell associated part. The blank reaction of a defined volume of specific culture fraction in 4 ml assay buffer was determined, and than the reaction was started through addition of 80 μl of substrate solution (100 mM PNP- butyrate in methanol), and the reaction was observed at 25°C at 400 nm in a spectrophotometer.

cell bound activity in laminarinase laminarinase activity* the medium extract extracted cells OD660 pSYl 0.001 (llόμl) pUR7034 0.293 (220μl) pUR2972B-(l) 0.494 (143μl)

* unless otherwise mentioned, the volume of enzyme solution added was 20μl

This result positively demonstrates that a significant amount of lipase activity is immobilized on the surface yeast cell, containing plasmid pUR2972B. Here again, incorporation took place in such a way, that the reaction was catalyzed by cell wall inserted lipase of intact cells, indicated into the exterior orientated immobilization. Furthermore, the release of a significant amount of lipase activity after incubation with laminarinase again demonstrates the presumably covalent incorporation of a heterologous enzyme through gene fusion with the C-terminal part of α-agglutinin. 3. Localization

The expression, secretion-, and subsequent incorporation of the lipase-α-agglutinin fusion protein into the yeast cell wall was also confirmed through immunofluorescent labelling with anti-lipolase serum essentially as described in EXAMPLE 1, item 2. Localization.

As can be seen in Figure 15, the immunofluorescent stain shows essentially an analogous picture as the α-galactosidase irnmuno stain, with clearly detectable reactivity on the outside of the cell surface (see Figure 15 A showing a clear halo around the cells and Figure B showing a lighter circle at the surface of the cells), but neither in the medium nor in the interior of the cells. Yeast cells expressing pUR2972B, the Humicola lipase-α-agglutinin fusion protein, become homogeneously stained on the surface, indicating the virtually entire immobilization of a chimeric enzyme with an α-agglutinin C-terminus on the exterior of a yeast cell. In the performed control experiment SU10 yeast cells containing plasmid pUR7034 served as a control and here, no cell surface bound reactivity against the applied anti-lipase serum could be detected.

In a similar way variants of Humicola lipase, obtained via rDNA techniques, can be linked to the C-terminal part of α-agglutinin, which variants can have a higher stability during (inter)esterification processes.

EXAMPLE 3 Immobilized Humicola lipase/α-agglutinin on the surface of S. cerevisiae (constitutive expression of immobilized enzyme system) Plasmid pUR2972 as described in EXAMPLE 2 can be treated with Eagl and H dlll and the about 2.2 kb fragment containing the lipase/α-agglutinin gene can be isolated. Plasmid pSYlό can be restricted with Eagl and Hindlll and between these sites the 2.2 kb fragment containing the lipase/α-agglutinin fragment can be ligated resulting in pUR2973. The part of this plasmid that is involved in the production of the chimeric enzyme is similar to pUR2972 with the exception of the signal sequence. Whereas pUR2972 contains the S£/C2-invertase-signal sequence, pUR2973 contains the α-mating factor signal sequence (see reference 18). Moreover the plasmid pUR2973 contains the Leu2 marker gene with the complete promoter sequence, instead of the truncated promoter version of pUR2972.

EXAMPLE 4 Immobilized Geotrichum lipase/α-agglutinin on the surface of S. cerevisiae

The construction and isolation of the 1.4 kb Nhel/HindlU fragment comprising the C-terminal part of AGα-1 (α-agglutinin) gene has been described in EXAMPLE 1. For the in-frame gene fusion of the DNA coding for the C-terminal membrane anchor of α-agglutinin to the complete coding sequence of Geotrichum candidum lipase B from strain CMICC 335426 (see Figure 8 and SEQ ID NO: 11 and 12), the plasmid pUR2974 can be used. This plasmid, derived from the commercially available pBluescript II SK plasmid, contains the cDNA coding for the complete G. candidum lipase II on an 1850 bp long EcoRl/Xfiol insert (see Figure 9). To develop an expression vector for S. cerevisiae with homologous signal sequences, the N-terminus of the mature lipase B was determined experimentally by standard techniques. The obtained amino acid sequence of "Gln-Ala-Pro-Thr-Ala-Val..." is in complete agreement with the cleavage site of the signal peptidase on the G. candidum lipase II (see reference 19).

For the fusion of the mature lipase B to the S. cerevisiae signal sequences of SUC2 (invertase) or α-mating factor (prepro-αMF) on one hand and the in-frame fusion to the 3' part of the AGαl gene PCR technique can be used. The PCR primer Hpo3 (see SEQ ID NO: 13) can be constructed in such a way, that the originally present Eagl site in the 5'-part of the coding sequence (spanning codons 5-7 of the mature protein) will become inactivated without any alteration in the amino acid sequence. To facilitate the subsequent cloning procedures, the PCR primer can further contain a new Eagl site at the 5' end, for the in-frame ligation to SUC2 signal sequence or prepro-αMF sequence, respectively. The corresponding PCR primer lipo4 (see SEQ ID NO: 16) contains an extra Mzel site behind the nucleotides coding for the C-terminus of lipase B, to ensure the proper fusion to the C-terminal part of α-agglutinin.

PCR oligonucleotides for the in frame linkage of G. candidum lipase II to the SUC2 signal sequence and the C-terminal part of α-agglutinin.

a: N-terminal transition to either prepro αMF sequence or SZ7C2 signal sequence.

Eagl A Q A P R P S N primer lipo3 : 5 ' -GGG GCG GCC GCG CAG GCC CCA AGG CGG TCT CTC AAT-3 ' lipasell: 3'-GAC CIGIG GITICI CIGIGI GIGITI GCICI GICICI AIGIAI GIAIGI TITIAI-5'

(non-cod. strand, see SEQ ID NO: 14) )

b: C-terminal fusion to C part of α-agglutinin

S N F E T D V N Y G lipase : 5 ' -CA AAC TTT GAG ACT GAC GTT AAT CTC TAC GGT TAA AAC-3 '

(cod. strand) | ||| ||| ||| ||| ||| III in primer lipo4: 3'-C TGA CTG CAA TTA GAG ATG CCA CGATCG CCCC-5'

Nhel (for the part of the lipase coding strand see SEQ ID NO: 15)

The PCR product with the modified ends can be generated by standard PCR protocols, using instead of the normal Ampli-7α<7 polymerase the new thermostable VENT polymerase, which also exhibits proofreading activity, to ensure an error-free DNA template. Through digestion of the formerly described plasmid pUR2972 with Eαgl (complete) and Nhel (partial), the Humicola lipase fragment can be exchanged against the DNA fragment coding for lipase B, thereby generating the final S. cerevisiae expression vector pUR2975 (see Figure 9).

The Humicola lipase-α-agglutinin fusion protein coding sequence can be exchanged against the lipase B/α-agglutinin fusion construct described above by digestion of the described vector ρUR2973 with Eagl/Hindlll, resulting in pUR2976 (see Figure 9).

EXAMPLE 5 Immobilized Rhizomucor miehei lipase/α-agglutinin on the surface of S. cerevisiae

The construction and isolation of the 1.4 kb Nhel/Hindlll fragment encoding the C-terminal part of α-agglutinin has been described in EXAMPLE 1. The plasmid pUR2980 contains a 1.25 kb cDNA fragment cloned into the Sm l site of commercially available pUClδ, which (synthetically synthesizable) fragment encodes the complete coding sequence of triglyceride lipase of Rhizomucor miehei (see reference 20), an enzyme used in a number of processes to interesterify triacylglycerols (see reference 21) or to prepare biosurfactants (see reference 22). Beside the 269 codons of the mature lipase molecule, the fragment also harbours codons for the 24 amino acid signal peptide as well as 70 amino acids of the propeptide. PCR can easily be applied to ensure the proper fusion of the gene frag¬ ment encoding the mature lipase to the SUC2 signal sequence or the prepro α-mating factor sequence of S. cerevisiae, as well as the in-frame fusion to the described Nhel/Hindlll fragment. The following two primers, lipo5 (see SEQ ID NO: 17) and liρo6 (see SEQ ID NO: 20), will generate a 833 bp DNA fragment, which after

Proteinase K treatment and digestion with Eagl and Nhel can be cloned as an 816 bp long fragment into the Eagl/Nhel digested plasmids pUR2972 and pUR2973, respectively (see Figure 7).

Eagl A S I D G G I lipo5: 5'-CCC GCG GCC GCG AGC ATT GAT GGT GGT ATC-3' lipase (non-cod. strand): 3*-T ICIGI TIAIAI CITIAI GICIAI CICIAI TIAIGI-5'

(for the part of the lipase non-coding strand see SEQ ID NO: 18) N T G C T lipase (cod. strand): δ'-AAC ACA GGC CTC TGT ACT-3'

Lipo6: 3'-T ITIGI TIGITI CICIGI GIAIGIAICIAI TIGIAI CGATCGCGCC-5'

Nhel (for the part of the lipase coding strand see SEQ ID NO: 19)

These new S. cerevisiae expression plasmids contain the GAL7 promoter, the invertase signal sequence (pUR2981) or the prepro-α-mating factor sequence (pUR2982), the chimeric Rhizomucor miehei lipase/α-agglutinin gene, the 2 μm sequence, the defective (truncated) Leu2 promoter and the Leu2 gene. These plasmids can be transformed into S. cerevisiae and grown and analyzed using protocols described in earlier EXAMPLES.

EXAMPLE 6 Immobilized Aspergillus niger glucose oxidase/GPI anchored cell wall proteins on the surface of S. cerevisiae

Glucose oxidase (β-D:oxygen 1-oxidoreductase, EC 1.1.3.4) from Aspergillus niger catalyses the oxidation of β-D-glucose to glucono-δ-lactone and the concomitant reduction of molecular oxygen to hydrogen peroxide. The fungal enzyme consists of a homodimer of molecular weight 150,000 containing two tightly bound FAD co-factors. Beside the use in glucose detection kits the enzyme is useful as a source of hydrogen peroxide in food preservation. The gene was cloned from both cDNA and genomic libraries, the single open reading frame contains no intervening sequences and encodes a protein of 605 amino acids (see reference 23). With the help of two proper oligonucleotides the coding part of the sequence is adjusted in a one-step modifying procedure by PCR in such a way that a fusion gene product will be obtained coding for glucose oxidase and the C-terminal cell wall anchor of the FLOl gene product or α-agglutinin. Thus, some of the plasmids described in former EXAMPLES can be utilized to integrate the corresponding sequence in-frame between one of the signal sequences used in the EXAMPLES and the Nhel/Hindlll part of the AGαl gene.

Since dimerisation of the two monomers might be a prerequisite for activity, in an alternative approach the complete coding sequence for glucose oxidase without the GPI anchor can be expressed in S. cerevisiae transformant which already contains the fusion construct. This can be fulfilled by constitutive expression of the fusion construct containing the GPI anchor with the help of the GAPDH or PGK promoter for example. The unbound not-anchored monomer can be produced by using a DNA construct comprising an inducible promoter, as for instance the GAL7 promoter.

EXAMPLE 7 Process to convert raffinose, stachyose and similar sugars in soy extracts with α-galactosidase/α-agglutinin immobilized on yeasts

The yeast transformed with plasmid pUR2969 can be cultivated on large scale. At regular intervals during cultivation the washed cells should be analyzed on the presence of α-galactosidase activity on their surface with methods described in EXAMPLE 1. When both cell density and α-galactosidase activity/biomass reach their maximum, the yeast cells can then be collected by centrifugation and washed. The washed cells can then be added to soy extracts. The final concentration of the yeast cells can vary between 0.1 and 10 g/1, preferably the concentration should be above 1 g/1. The temperature of the soy extract should be < 8 °C to reduce the metabolic activity of the yeast cells. The conversion of raffinose and stachyose can be analyzed with HPLC methods and after 95 % conversion of these sugars the yeasts cells can be removed by centrifugation and their α-galactosidase activity /g biomass can be measured. Centrifugates with a good activity can be used in a subsequent conversion process, whereas centrifugates with an activity of less then 50 % of the original activity can be resuscitated in the growth medium and the cells can be allowed to recover for 2 to 4 hours. Thereafter the cells can be centrifuged, washed and subsequently be used in a subsequent conversion process.

EXAMPLE 8 Production of biosurfactants using Humicola lipase/α-agglutinin immobilized on yeasts. The yeast transformed with plasmid pUR2972 or pUR2973 can be cultivated on large scale. At regular intervals during cultivation the washed cells can be analyzed on the presence of lipase activity on their surface with methods described in EXAMPLE 1. When both cell density and lipase/biomass reache their maximum, the yeast cells can be collected by centrifugation and washed. The washed cells can be suspended in a small amount of water and added to a reactor tank containing a mix of fatty acids, preferably of a chain length between 12-18 carbon atoms and sugars, preferably glucose, galactose or sucrose. The total concentration of the water (excluding the water in the yeast cells) might be below 0.1 %. The final concentration of the yeast cells can vary between 0.1 and 10 g/1, preferably the concentration is above 1 g/1. The tank has to be kept under an atmosphere of N₂ and CO₂ in order to avoid oxidation of the (unsaturated) fatty acids and to minimize the metabolic activity of the yeasts. The temperature of mixture in the tank should be between 30-60 °C, depending on type of fatty acid used. The conversion of fatty acids can be analyzed with GLC methods and after 95 % conversion of these fatty acids the yeasts cells can be removed by centrifugation and their lipase activity /g biomass can be measured. Centrifugates with a good activity can be used in a subsequent conversion process, whereas centrifugates with an activity of less then 50 % of the original activity can be resuscitated in the growth medium and the cells can be allowed to recover for 2 to 8 hours. Thereafter the cells can be centrifuged again, washed and used in a subsequent conversion process. EXAMPLE 9 Production of special types of triacylglycerols using Rhizomucor miehei lipase/α-agglutinin immobilized on yeasts.

The yeast transformed with plasmid pUR2981 or pUR2982 can be cultivated on a large scale. At regular intervals during cultivation the washed cells can be analyzed on the presence of lipase activity on their surface with methods described in EXAMPLE 1. When both cell density and lipase/biomass reach their maximum, the yeast cells can be collected by centrifugation and washed. The washed cells can be suspended in a small amount of water and can be added to a reactor tank containing a mix of various triacylglycerols and fatty acids. The total concentration of the water (excluding the water in the yeast cells) might be below 0.1 %. The final concentration of the yeast cells can vary between 0.1 and 10 g/1, preferably the concentration is above 1 g/1. The tank has to be kept under an atmosphere of N₂ and CO₂ in order to avoid oxidation of the (unsaturated) fatty acids and to minimize the metabolic activity of the yeasts. The temperature of mixture in the tank should be between 30-70 °C, depending on types of triacylglycerol and fatty acid used. The degree of interesteri- fication can be analyzed with GLC/MS methods and after formation of at least 80 % of the theoretical value of the desired type of triacylglycerol the yeasts cells can be removed by centrifugation and their lipase activity /g biomass can be measured. Centrifugates with a good activity can be used in a subsequent conversion process, whereas centrifugates with an activity of less then 50 % of the original activity is resuscitated in the growth medium and the cells should be allowed to recover 2 to 8 hours. After that the cells can be centrifuged, washed and used in a subsequent inter- esterification process. Baker's yeasts of strain MT302/1C, transformed with either plasmid pSY13 or plasmid pUR2969 (described in EXAMPLE 1) were deposited under the Budapest Treaty at the Centraalbureau voor Schimmelcultures (CBS) on 3 July 1992 under provisional numbers 330.92 and 329.92, respectively.

EXAMPLE 10 Immobilized Humicola lipase/FLOl fusion on the surface of S. cerevisiae

Flocculation, defined as "the (reversible) aggregation of dispersed yeast cells into floes" (see reference 24), is the most important feature of yeast strains in industrial fermentations. Beside this it is of principal interest, because it is a property associated with cell wall proteins and it is a quantitative characteristic. One of the genes associated with the flocculation phenotype in S. cerevisiae is the FLOl gene. The gene is located at approximately 24 kb from the right end of chromosome I and the DNA sequence of a clone containing major parts of FLOl gene has very recently been determined (see reference 26). The sequence is given in Figure 11 and SEQ ID NO: 21 and 22. The cloned fragment appeared to be approximately 2 kb shorter than the genomic copy as judged from Southern and Northern hybridizations, but encloses both ends of the FLOl gene. Analysis of the DNA sequence data indicates that the putative protein contains at the N-terminus a hydrophobic region which confirms a signal sequence for secretion, a hydrophobic C-terminus that might function as a signal for the attachment of a GPI-anchor and many glycosylation sites, especially in the C-terminus, with 46,6 % serine and threonine in the arbitrarily defined C-termi¬ nus (aa 271-894). Hence, it is likely that the FLOl gene product is localized in an orientated fashion in the yeast cell wall and may be directly involved in the process of interaction with neighbouring cells. The cloned FLOl sequence might therefore be suitable for the immobilization of proteins or peptides on the cell surface by a dif¬ ferent type of cell wall anchor. Recombinant DNA constructs can be obtained, for example by utilizing the DNA coding for amino acids 271-894 of the FLOl gene product, i.e. polynucleotide

811-2682 of Figure 11. Through application of two PCR primers pcrflol (see SEQ ID NO: 23) and pcrflo2 (see SEQ ID NO: 26) Mzel and Hindlll sites can be introduced at both ends of the DNA fragment. In a second step, the 1.4 kb Nhel/Hindlll fragment present in pUR2972 (either A or B) containing the C-terminal part of α-agglutinin can be replaced by the 1.9 kb DNA fragment coding for the C-terminal part of the FLOl protein, resulting in plasmid pUR2990 (see Figure 12), comprising a DNA sequence encoding (a) the invertase signal sequence (SUC2) preceding (b) the fusion protein consisting of (b. ) the lipase of Humicola (see reference 16) followed by (b.2) the C-terminus of FLOl protein (aa 271-894). PCR oligonucleotides for the in frame connection of the genes encoding the Humicola lipase and the C-terminal part of the FLOl gene product.

S N Y A V S T primer pcrflol 5'- GAATTC GCT AGC AAT TAT GCT GTC AGT ACC - 3'

FLOl gene (non-coding strand) M3'e-l—AGT THTAI AITIAI CIGIAI CIAIGI TICIAI TIGIGI TGA - 5' (for the part of the non-coding strand see SEQ ID NO: 24)

FLOl coding Strand 5'-AATAA AATTCGCGTTCTTTTTACG - 3' primer pcrflo2: 3'-TlTlAlAlGlClGlClAlAlGlAlAlAlAlAlTlGlClTTCGAACTCGAG - 5'

Hindlll (for the part of the coding strand see SEQ ID NO: 25)

Plasmid pUR2972 (either A or B) can be restricted with Nhel (partial) and Htndlll and the Nhel/Hindlll fragment comprising the vector backbone and the lipase gene can be ligated to the correspondingly digested PCR product of the plasmid containing the FLOl sequence, resulting in plasmid pUR2990, containing the GAL7 promoter, the S. cerevisiae invertase signal sequence, the chimeric lipase/FLOJf gene, the yeast 2 μm sequence, the defective Leu2 promoter and the Leu2 gene. This plasmid can be transformed into S. cerevisiae and the transformed cells can be cultivated in YP medium including galactose as inductor. The expression, secretion, localization and activity of the chimeric lipase/FLOl protein can be analyzed using similar procedures as given in Example 1.

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25. Schreuder, M.P., Brekelmans, S., Van den Ende, H., Klis, F.M. (1993) 'Targeting of a Heterologous Protein to the Cell Wall of Saccharomyces cerevisiae" Yeast 9 399-409

26. Teunissen, A.W.R.H., Holub, E., Van der Hucht, J., Van Den Berg, J.A., Steensma, H.Y. (1993) "Sequence of the Open reading frame of the FLOl Gene from Saccharomyces cerevisiae" YEAST 9 423-427.

27. Harmsen, M.M., Langedijk, A.C., Van Tuinen, E., Geerse, R.H.; Raue, H.A., Maat, J. (1993) Effect of ά ' pmrl disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba α-galactosidase by Saccharomyces cerevisiae Gene 125 115-123 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

NAME: Unilever N.V. STREET: Weena 455 CITY: Rotterdam COUNTRY: The Netherlands POSTAL CODE (ZIP): NL-3013 AL

NAME: Unilever PLC

STREET: Unilever House Blackfriars

CITY: London

COUNTRY: United Kingdom

POSTAL CODE (ZIP): EC4P 4BQ

NAME: Franciscus Maria KLIS STREET: Benedenlangs 102 CITY: Amsterdam COUNTRY: The Netherlands POSTAL CODE (ZIP): NL-1025 KL

NAME: Maarten Pleun SCHREUDER STREET: Rode Kruislaan 1220 CITY: Diemen COUNTRY: The Netherlands POSTAL CODE (ZIP): NL-1111 XB

NAME: Holger York TOSCHKA STREET: Coornhertstraat 77 CITY: Vlaardingen COUNTRY: The Netherlands POSTAL CODE (ZIP): NL-3132 GB

NAME: Cornelis Theodorus VERRIPS STREET: Hagedoorn 18 CITY: Maassluis COUNTRY: The Netherlands POSTAL CODE (ZIP): NL-3142 KB

(ii) TITLE OF INVENTION: Enzymic Processes based on naturally immobilized enzymes that can easily be separated and regenerated (iii) NUMBER OF SEQUENCES: 26

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6057 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Saccharomyces cerevisiae

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 3653..5605

(D) OTHER INFORMATION: /function= "sexual agglutinisation" /product= "alpha-agglutinin"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

AAGCTTTAGG TAAGGGAGGC AGGGGGAAAA GATACTGAAA TGACGGAAAA CGAGAATATG 60

GAGCAGGGAG CAACTTTTAG AGCTTTACCC GTTAAAAGGT CAAATCGAGG CTTCCTGCCT 120

TTGTCTGATT TTAGTAGTAC CGGAAGGTTT ATTACGCCCA AGAACAGTGC TTGAATTGAG 180

TTCTCGGGAC ACGGGAAAGA CAATGGAAGA AAAATTTACA TTCAGTAGCC TTATATATGA 240

AATGCTGCCA AGCCACGTCT TTATAAGTAG ATAATGTCCC ATGAGCTGAA CTATGGGAAT 300

TTATGACGCA GTTCATTGTA TATATATTAC ATTAACTCTT TAGTTTAACA TCTGAATTGT 360

TTTATAAAAT AACTTTTTGA ATTTTTTTAT GATCGCTTAG TTAAGTCTAT TATATCAGGT 420

TTTTTCATTC ATCATAATTG TTCGTTAAAT ATGAGTATAT TTAAATACAG GAATTAGTAT 480 CATTTGCAGT CACGAAAAGG GCCGTTTCAT AGAGAGTTTT CTTAATAAAG TTGAGGGTTT 540

CCGTGATAGT TTTGAGGGGT TGTTTGAACT AGATTTACGC TTACCTTTCA ACTGATTAAT 600

TTTTTCAGCG GGCTTATCAT AATCATCCAT CATAGCAGTC TTTCTGGACT TCGTCGAGGA 660

CTGGCTTTCT GAATTTTGAC GGTCCCTATT AGCTCCAGTT GGAGGAATTG AGTTACCTAC 720

AACTGGCAAG AGGTCTTTGT TTGGATTCAA AATAGGACTT TGTGGTAGCA GTTTGGTTTT 780

ATTCAATCTA AAGATATGAG AAACAGGTTT TAAGTAAATC GATACTATTG TACCAATGTT 840

TAGCTCCAAT TCCTCCAAAA CGGTGGGATC TAATTTTGTG TTCATTTCTA TTAGTGGCAA 900

CTCTCCGTCC AGTACTGATT TTAAAGATTC AAAAGTTATC GCGTTTGATA TACGAGACGT 960

TTTCGTTAAT GACAGCAATC TCCAATACAT CAGTGTTTTA TCTCTTAAGT CAGGATTATT 1020

TTCGTGATCG GTGCATCCTT TTAATAAATC CATACAAAGT TCTTCAGTTT CCTTTGTAGG 1080

ATTTCTGATG AAGAATTTTA TTGCTGAGTT CAGAATGGAA AATTGCACTT CTAGCGTCTC 1140

ATTAAACATG TTTGAGGAAA AAACTCTAAA TAACTCCAGG TAGTTTGGAA TTACATCCGA 1200

ATATTGCGTT ATTATCCAGA TCATAGCGTT TTTTGATTCA GGTTCCTGTA CAACTTCAGT 1260

GTGTTTGACT AGTTCTGTTA CGTTTGCTTT AAAATTATTG GGATATTTCC TCAAAATATT 1320

TCTGAAAACC GAAATAATCT CCTGGACGAC ATAATCAACA CCGAATTCTA ACAAATCTAG 1380

TAGCACAGCG ACACAATCGT GTACAGAGTC TTCATCTAGC TTAACAGCGA GATTACCAAT 1440

GGCTCTGACT GATTTCCTTG ACATTTGAAT ATCAATATCT GTAGCATATT GTTCCAACTC 1500

TTCTAGAATT CTTGGTAATG TTTCCTTGTT AGCTAAAAGA TATAAACACT CTAATTTCGT 1560

GTCTTTGATG TATATGGGGT CATTGTACTC GATGAAAAAA TACGAAATGT CTAGCCTGAG 1620

TAGAGATGAC TCCCTACTCA ATAAAAGAAG AATAACGTTT CTTAATACTA AAAATTGTAA 1680

TTCAGGCGGC TTATCTAACA AAGCTATTAC AGAGTTAGAT AGCTTTTCGG CTAGAGTTTC 1740

TTTGATGACG TCAACATAAT TCAACAAGTA CATGATGAAT TTTAAAGAGT TCAACACTAC 1800

GTATGTGTTT ACTTGTTGCA GGTACGGTAA AGCTAGTTCG ATCATTTCAT GGGTATCCAA 1860 ATAATGCTGC GGCACAACCG AAGTCGTCAA AACTTCCAAA ACAGTAGCCT TATTCCACTC 1920

ATTTAATTCG GGTAAAAGTT CTAGCATGTC AAAAGCGAGT TCCAAGGGAA TCCTGAAGGT 1980

TCCATGTTAG CGTTTTTTTC GTGAATGGAA TATAAAGTAT GTAATGCAGC TACAATGACT 2040

TCTGGAGAGC TCGACTGTGC CTTTACAATG TCATGTAGAA TGCTTGATAA CCCCAATACC 2100

CTTTCATGAT CAATTTCATC TAAATCCAAC AGTGCGTAAA TTGCTGTCCT CGTCACTTGT 2160

TCAGGTGGAG ACTTGTGATT TACCAATGAA ATGATACAGT CGAAGGCCTG ATCAGATAGC 2220

TCTTTCACCG GGACTAATAC CAGAGTTCTT AGTGCCATTA TTTGTAACTT TTCATCTCTG 2280

CTTTTGAAAT CGTCCATTAT AAATGGCAAA GCCTCTCTGG CCTGCTGAGG TTTTAATGCG 2340

CCGATCACCC TAATATACTC ATGGCAAATT CTTTTCACTT CTAGATCATC TTCAATTTGC 2400

CAAAATTTCA AGAGCTCAGA AAACAGAAGG GACATTTCGC CATAGTTTCC TAGAACCAAA 2460

TTGGCGATAA TTTTTCTCAG AGCATTTTTC CTTCTTGTTA TATTCGATTT AAACTTTTTT 2520

ACTCCAAAAT GTTGCAGATC TGTGACGATT TCATTTGCTT TATATCTGGC AAAAACTTTT 2580

TGATCGGACA TAAGCGAAAT ACGTCCTATT AATGAAGTGA ATGTTCTTGC TGTATTCCCT 2640

TCTTGTGCAG TAGATTAATT CTGTTTCCAG GCTGCGATAC TTTGATACCC AATACTAAAA 2700

GTTGATGATT TGAACGATCT CCTATTTCCT CGCACATTTT TGGAGCGATA CCCGGAAGAC 2760

AGAATCGCGA TGTTAAGAAA ATAGTTCTGA TGGCACTAAA GAGATCATGA TTAAGGAAAG 2820

GTAAGTGATA TGCATGAATG GGAATAGGCT TTCGAACTTG ACGATTTAGT TCCTTATTTC 2880

TATCCATCTA ATCCTCCAAC TTCAATAGGC CTTATCTAGC TCAGAGCAGT ATTTAATTGA 2940

GAATAGTAGC TTAATTGAAA CCTTACTAAA AAAGTGTATG GTTACATAAG ATAAGGCGTT 3000

AAGAAGAGTA TACATATGCA TTATTCATTA CCAAGACCAC TATGAATAGT AATACCATAT 3060

TTAGCTTTTG AAACTCATGT TTTCTATTGT GTTGTTTCAA ATTCCTCTGT TAGGCTCAAT 3120

TTAGGTTAAT TAAATTATAA AAAAATATAA AAAATAAAGA AAGTTTATCC ATCGGCACCT 3180

CAATTCAATG GAGTAAACAG TTTCAACACT GAGTGGTGAA ACATTGAACA ACTACATGCA 3240 GTTTCCCGCC ACGAGGCAAG TGTAGGTCCT TTGTCCATTT CGCTTTGTTT TGCAGGTCAT 3300

TGATGACCTA ATTAGGAAGG TAGAAGCCGC TCCAGCTCAA TAAGGAAATG CTAAGGGTAC 3360

TCGCCTTTGG TGTTTTACCA TACAATGGCA GCTTTATGTC ACTTCATTCT TCAGTAACGG 3420

CGCTTAAATA TTCCCAAAAA CGTTACAATG GAATTGTTTG ATCATGTAAC GAAATGCAAT 3480

CTTCTAAAAA AAAAGCCATG TGAATCAAAA AAAGATTCCT TTTAGCATAC TATAAATATG 3540

CAAAATGCCC TCTATTTATT CTAGTAATCG TCCATTCTCA TATCTTCCTT ATATCAGTCG 3600

CCTCGCTTAA TATAGTCAGC ACAAAAGGAA CAACAATTCG CCAGTTTTCA AA ATG 3655

Met

1

TTC ACT TTT CTC AAA ATT ATT CTG TGG CTT TTT TCC TTG GCA TTG GCC 3703 Phe Thr Phe Leu Lys lie lie Leu Trp Leu Phe Ser Leu Ala Leu Ala 5 10 15

TCT GCT ATA AAT ATC AAC GAT ATC ACA TTT TCC AAT TTA GAA ATT ACT 3751 Ser Ala lie Asn lie Asn Asp lie Thr Phe Ser Asn Leu Glu lie Thr 20 25 30

CCA CTG ACT GCA AAT AAA CAA CCT GAT CAA GGT TGG ACT GCC ACT TTT 3799 Pro Leu Thr Ala Asn Lys Gin Pro Asp Gin Gly Trp Thr Ala Thr Phe 35 40 45

GAT TTT AGT ATT GCA GAT GCG TCT TCC ATT AGG GAG GGC GAT GAA TTC 3847 Asp Phe Ser lie Ala Asp Ala Ser Ser lie Arg Glu Gly Asp Glu Phe 50 55 60 65

ACA TTA TCA ATG CCA CAT GTT TAT AGG ATT AAG CTA TTA AAC TCA TCG 3895 Thr Leu Ser Met Pro His Val Tyr Arg lie Lys Leu Leu Asn Ser Ser 70 75 80

CAA ACA GCT ACT ATT TCC TTA GCG GAT GGT ACT GAG GCT TTC AAA TGC 3943 Gin Thr Ala Thr He Ser Leu Ala Asp Gly Thr Glu Ala Phe Lys Cys 85 90 95

TAT GTT TCG CAA CAG GCT GCA TAC TTG TAT GAA AAT ACT ACT TTC ACA 3991 Tyr Val Ser Gin Gin Ala Ala Tyr Leu Tyr Glu Asn Thr Thr Phe Thr 100 105 110 TGT ACT GCT CAA AAT GAC CTG TCC TCC TAT AAT ACG ATT GAT GGA TCC 4039 Cys Thr Ala Gin Asn Asp Leu Ser Ser Tyr Asn Thr He Asp Gly Ser 115 120 125

ATA ACA TTT TCG CTA AAT TTT AGT GAT GGT GGT TCC AGC TAT GAA TAT 4087 He Thr Phe Ser Leu Asn Phe Ser Asp Gly Gly Ser Ser Tyr Glu Tyr 130 135 140 145

GAG TTA GAA AAC GCT AAG TTT TTC AAA TCT GGG CCA ATG CTT GTT AAA 4135 Glu Leu Glu Asn Ala Lys Phe Phe Lys Ser Gly Pro Met Leu Val Lys 150 155 160

CTT GGT AAT CAA ATG TCA GAT GTG GTG AAT TTC GAT CCT GCT GCT TTT 4183 Leu Gly Asn Gin Met Ser Asp Val Val Asn Phe Asp Pro Ala Ala Phe 165 170 175

ACA GAG AAT GTT TTT CAC TCT GGG CGT TCA ACT GGT TAC GGT TCT TTT 4231 Thr Glu Asn Val Phe His Ser Gly Arg Ser Thr Gly Tyr Gly Ser Phe 180 185 190

GAA AGT TAT CAT TTG GGT ATG TAT TGT CCA AAC GGA TAT TTC CTG GGT 4279 Glu Ser Tyr His Leu Gly Met Tyr Cys Pro Asn Gly Tyr Phe Leu Gly 195 200 205

GGT ACT GAG AAG ATT GAT TAC GAC AGT TCC AAT AAC AAT GTC GAT TTG 4327 Gly Thr Glu Lys He Asp Tyr Asp Ser Ser Asn Asn Asn Val Asp Leu 210 215 220 225

GAT TGT TCT TCA GTT CAG GTT TAT TCA TCC AAT GAT TTT AAT GAT TGG 4375 Asp Cys Ser Ser Val Gin Val Tyr Ser Ser Asn Asp Phe Asn Asp Trp 230 235 240

TGG TTC CCG CAA AGT TAC AAT GAT ACC AAT GCT GAC GTC ACT TGT TTT 4423 Trp Phe Pro Gin Ser Tyr Asn Asp Thr Asn Ala Asp Val Thr Cys Phe 245 250 255

GGT AGT AAT CTG TGG ATT ACA CTT GAC GAA AAA CTA TAT GAT GGG GAA 4471 Gly Ser Asn Leu Trp He Thr Leu Asp Glu Lys Leu Tyr Asp Gly Glu 260 265 270

ATG TTA TGG GTT AAT GCA TTA CAA TCT CTA CCC GCT AAT GTA AAC ACA 4519 Met Leu Trp Val Asn Ala Leu Gin Ser Leu Pro Ala Asn Val Asn Thr 275 280 285 ATA GAT CAT GCG TTA GAA TTT CAA TAC ACA TGC CTT GAT ACC ATA GCA 4567 He Asp His Ala Leu Glu Phe Gin Tyr Thr Cys Leu Asp Thr He Ala 290 295 300 305

AAT ACT ACG TAC GCT ACG CAA TTC TCG ACT ACT AGG GAA TTT ATT GTT 4615 Asn Thr Thr Tyr Ala Thr Gin Phe Ser Thr Thr Arg Glu Phe He Val 310 315 320

TAT CAG GGT CGG AAC CTC GGT ACA GCT AGC GCC AAA AGC TCT TTT ATC 4663 Tyr Gin Gly Arg Asn Leu Gly Thr Ala Ser Ala Lys Ser Ser Phe He 325 330 335

TCA ACC ACT ACT ACT GAT TTA ACA AGT ATA AAC ACT AGT GCG TAT TCC 4711 Ser Thr Thr Thr Thr Asp Leu Thr Ser He Asn Thr Ser Ala Tyr Ser 340 345 350

ACT GGA TCC ATT TCC ACA GTA GAA ACA GGC AAT CGA ACT ACA TCA GAA 4759 Thr Gly Ser He Ser Thr Val Glu Thr Gly Asn Arg Thr Thr Ser Glu 355 360 365

GTG ATC AGT CAT GTG GTG ACT ACC AGC ACA AAA CTG TCT CCA ACT GCT 4807 Val He Ser His Val Val Thr Thr Ser Thr Lys Leu Ser Pro Thr Ala 370 375 380 385

ACT ACC AGC CTG ACA ATT GCA CAA ACC AGT ATC TAT TCT ACT GAC TCA 4855 Thr Thr Ser Leu Thr He Ala Gin Thr Ser He Tyr Ser Thr Asp Ser 390 395 400

AAT ATC ACA GTA GGA ACA GAT ATT CAC ACC ACA TCA GAA GTG ATT AGT 4903 Asn He Thr Val Gly Thr Asp He His Thr Thr Ser Glu Val He Ser 405 410 415

GAT GTG GAA ACC ATT AGC AGA GAA ACA GCT TCG ACC GTT GTA GCC GCT 4951 Asp Val Glu Thr He Ser Arg Glu Thr Ala Ser Thr Val Val Ala Ala 420 425 430

CCA ACC TCA ACA ACT GGA TGG ACA GGC GCT ATG AAT ACT TAC ATC CCG 4999 Pro Thr Ser Thr Thr Gly Trp Thr Gly Ala Met Asn Thr Tyr He Pro 435 440 445

CAA TTT ACA TCC TCT TCT TTC GCA ACA ATC AAC AGC ACA CCA ATA ATC 5047 Gin Phe Thr Ser Ser Ser Phe Ala Thr He Asn Ser Thr Pro He He 450 455 460 465 TCT TCA TCA GCA GTA TTT GAA ACC TCA GAT GCT TCA ATT GTC AAT GTG 5095 Ser Ser Ser Ala Val Phe Glu Thr Ser Asp Ala Ser He Val Asn Val 470 475 480

CAC ACT GAA AAT ATC ACG AAT ACT GCT GCT GTT CCA TCT GAA GAG CCC 5143 His Thr Glu Asn He Thr Asn Thr Ala Ala Val Pro Ser Glu Glu Pro 485 490 495

ACT TTT GTA AAT GCC ACG AGA AAC TCC TTA AAT TCC TTC TGC AGC AGC 5191 Thr Phe Val Asn Ala Thr Arg Asn Ser Leu Asn Ser Phe Cys Ser Ser 500 505 510

AAA CAG CCA TCC AGT CCC TCA TCT TAT ACG TCT TCC CCA CTC GTA TCG 5239 Lys Gin Pro Ser Ser Pro Ser Ser Tyr Thr Ser Ser Pro Leu Val Ser 515 520 525

TCC CTC TCC GTA AGC AAA ACA TTA CTA AGC ACC AGT TTT ACG CCT TCT 5287 Ser Leu Ser Val Ser Lys Thr Leu Leu Ser Thr Ser Phe Thr Pro Ser 530 535 540 545

GTG CCA ACA TCT AAT ACA TAT ATC AAA ACG GAA AAT ACG GGT TAC TTT 5335 Val Pro Thr Ser Asn Thr Tyr He Lys Thr Glu Asn Thr Gly Tyr Phe 550 555 560

GAG CAC ACG GCT TTG ACA ACA TCT TCA GTT GGC CTT AAT TCT TTT AGT 5383 Glu His Thr Ala Leu Thr Thr Ser Ser Val Gly Leu Asn Ser Phe Ser 565 570 575

GAA ACA GCA CTC TCA TCT CAG GGA ACG AAA ATT GAC ACC TTT TTA GTG 5431 Glu Thr Ala Leu Ser Ser Gin Gly Thr Lys He Asp Thr Phe Leu Val 580 585 590

TCA TCC TTG ATC GCA TAT CCT TCT TCT GCA TCA GGA AGC CAA TTG TCC 5479 Ser Ser Leu He Ala Tyr Pro Ser Ser Ala Ser Gly Ser Gin Leu Ser 595 600 605

GGT ATC CAA CAG AAT TTC ACA TCA ACT TCT CTC ATG ATT TCA ACC TAT 5527 Gly He Gin Gin Asn Phe Thr Ser Thr Ser Leu Met He Ser Thr Tyr 610 615 620 625

GAA GGT AAA GCG TCT ATA TTT TTC TCA GCT GAG CTC GGT TCG ATC ATT 5575 Glu Gly Lys Ala Ser He Phe Phe Ser Ala Glu Leu Gly Ser He He 630 635 640 TTT CTG CTT TTG TCG TAC CTG CTA TTC TAAAACGGGT ACTGTACAGT 5622

Phe Leu Leu Leu Ser Tyr Leu Leu Phe 645 650

TAGTACATTG AGTCGAAATA TACGAAATTA TTGTTCATAA TTTTCATCCT GGCTCTTTTT 5682

TTCTTCAACC ATAGTTAAAT GGACAGTTCA TATCTTAAAC TCTAATAATA CTTTTCTAGT 5742

TCTTATCCTT TTCCGTCTCA CCGCAGATTT TATCATAGTA TTAAATTTAT ATTTTGTTCG 5802

TAAAAAGAAA AATTTGTGAG CGTTACCGCT CGTTTCATTA CCCGAAGGCT GTTTCAGTAG 5862

ACCACTGATT AAGTAAGTAG ATGAAAAAAT TTCATCACCA TGAAAGAGTT CGATGAGAGC 5922

TACTTTTTCA AATGCTTAAC AGCTAACCGC CATTCAATAA TGTTACGTTC TCTTCATTCT 5982

GCGGCTACGT TATCTAACAA GAGGTTTTAC TCTCTCATAT CTCATTCAAA TAGAAAGAAC 6042

ATAATCAAAA AGCTT 6057

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 650 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Phe Thr Phe Leu Lys He He Leu Trp Leu Phe Ser Leu Ala Leu 1 5 10 15

Ala Ser Ala He Asn He Asn Asp He Thr Phe Ser Asn Leu Glu He 20 25 30

Thr Pro Leu Thr Ala Asn Lys Gin Pro Asp Gin Gly Trp Thr Ala Thr 35 40 45

Phe Asp Phe Ser He Ala Asp Ala Ser Ser He Arg Glu Gly Asp Glu 50 55 60 Phe Thr Leu Ser Met Pro His Val Tyr Arg He Lys Leu Leu Asn Ser 65 70 75 80

Ser Gin Thr Ala Thr He Ser Leu Ala Asp Gly Thr Glu Ala Phe Lys 85 90 95

Cys Tyr Val Ser Gin Gin Ala Ala Tyr Leu Tyr Glu Asn Thr Thr Phe 100 105 110

Thr Cys Thr Ala Gin Asn Asp Leu Ser Ser Tyr Asn Thr He Asp Gly 115 120 125

Ser He Thr Phe Ser Leu Asn Phe Ser Asp Gly Gly Ser Ser Tyr Glu 130 135 140

Tyr Glu Leu Glu Asn Ala Lys Phe Phe Lys Ser Gly Pro Met Leu Val 145 150 155 160

Lys Leu Gly Asn Gin Met Ser Asp Val Val Asn Phe Asp Pro Ala Ala 165 170 175

Phe Thr Glu Asn Val Phe His Ser Gly Arg Ser Thr Gly Tyr Gly Ser 180 185 190

Phe Glu Ser Tyr His Leu Gly Met Tyr Cys Pro Asn Gly Tyr Phe Leu 195 200 205

Gly Gly Thr Glu Lys He Asp Tyr Asp Ser Ser Asn Asn Asn Val Asp 210 . 215 220

Leu Asp Cys Ser Ser Val Gin Val Tyr Ser Ser Asn Asp Phe Asn Asp 225 230 235 240

Trp Trp Phe Pro Gin Ser Tyr Asn Asp Thr Asn Ala Asp Val Thr Cys 245 250 255

Phe Gly Ser Asn Leu Trp He Thr Leu Asp Glu Lys Leu Tyr Asp Gly 260 265 270

Glu Met Leu Trp Val Asn Ala Leu Gin Ser Leu Pro Ala Asn Val Asn 275 280 285

Thr He Asp His Ala Leu Glu Phe Gin Tyr Thr Cys Leu Asp Thr He 290 295 300 Ala Asn Thr Thr Tyr Ala Thr Gin Phe Ser Thr Thr Arg Glu Phe He 305 310 315 320

Val Tyr Gin Gly Arg Asn Leu Gly Thr Ala Ser Ala Lys Ser Ser Phe 325 330 335

He Ser Thr Thr Thr Thr Asp Leu Thr Ser He Asn Thr Ser Ala Tyr 340 345 350

Ser Thr Gly Ser He Ser Thr Val Glu Thr Gly Asn Arg Thr Thr Ser 355 360 365

Glu Val He Ser His Val Val Thr Thr Ser Thr Lys Leu Ser Pro Thr 370 375 380

Ala Thr Thr Ser Leu Thr He Ala Gin Thr Ser He Tyr Ser Thr Asp 385 390 395 400

Ser Asn He Thr Val Gly Thr Asp He His Thr Thr Ser Glu Val He 405 410 415

Ser Asp Val Glu Thr He Ser Arg Glu Thr Ala Ser Thr Val Val Ala 420 425 430

Ala Pro Thr Ser Thr Thr Gly Trp Thr Gly Ala Met Asn Thr Tyr He 435 440 445

Pro Gin Phe Thr Ser Ser Ser Phe Ala Thr He Asn Ser Thr Pro He 450 455 460

He Ser Ser Ser Ala Val Phe Glu Thr Ser Asp Ala Ser He Val Asn 465 470 475 480

Val His Thr Glu Asn He Thr Asn Thr Ala Ala Val Pro Ser Glu Glu 485 490 495

Pro Thr Phe Val Asn Ala Thr Arg Asn Ser Leu Asn Ser Phe Cys Ser 500 505 510

Ser Lys Gin Pro Ser Ser Pro Ser Ser Tyr Thr Ser Ser Pro Leu Val 515 520 525

Ser Ser Leu Ser Val Ser Lys Thr Leu Leu Ser Thr Ser Phe Thr Pro 530 535 540 Ser Val Pro Thr Ser Asn Thr Tyr He Lys Thr Glu Asn Thr Gly Tyr 545 550 555 560

Phe Glu His Thr Ala Leu Thr Thr Ser Ser Val Gly Leu Asn Ser Phe 565 570 575

Ser Glu Thr Ala Leu Ser Ser Gin Gly Thr Lys He Asp Thr Phe Leu 580 585 590

Val Ser Ser Leu He Ala Tyr Pro Ser Ser Ala Ser Gly Ser Gin Leu 595 600 605

Ser Gly He Gin Gin Asn Phe Thr Ser Thr Ser Leu Met He Ser Thr 610 615 620

Tyr Glu Gly Lys Ala Ser He Phe Phe Ser Ala Glu Leu Gly Ser He 625 630 635 640

He Phe Leu Leu Leu Ser Tyr Leu Leu Phe 645 650

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: primer lipol

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GGGGCGGCCG AGGTCTCGCA AGATCTGGA 29 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Part non-coding strand lipase

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

TTTGTCCAGG TCTTGCGAGA CCTCTCGACG AAT 33

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Part coding strand lipase

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TTCGGGTTAA TTGGGACATG TCTTTAGTGC GA 32

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:

(B) CLONE: primer lipo2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CCCCAAGCTT AAGGCTAGCA AGACATGTCC CAATTAACCC 40

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 894 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Humicola lanug±nosa

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 72..884

(D) OTHER INFORMATION: /product= "lipase"

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 72..881

(D) OTHER INFORMATION: /product= "lipase"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GAATTCGTAG CGACGATATG AGGAGCTCCC TTGTGCTGTT CTTTGTCTCT GCGTGGACGG 60

CCTTGGCCAC G GCC GAG GTC TCG CAA GAT CTG TTT AAC CAG TTC AAT CTC 110 Ala Glu Val Ser Gin Asp Leu Phe Asn Gin Phe Asn Leu

1 5 10

TTT GCA CAG TAT TCT GCT GCC GCA TAC TGC GGA AAA AAC AAT GAT GCC 158 Phe Ala Gin Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala 15 20 25 CCA GCT GGT ACA AAC ATT ACG TGC ACG GGA AAT GCC TGC CCC GAG GTA 206 Pro Ala Gly Thr Asn He Thr Cys Thr Gly Asn Ala Cys Pro Glu Val 30 35 40 45

GAG AAG GCG GAT GCA ACG TTT CTC TAC TCG TTT GAA GAC TCT GGA GTG 254 Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val 50 55 60

GGC GAT GTC ACC GGC TTC CTT GCT CTA GAC AAC ACG AAC AAA TTG ATC 302 Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu He 65 70 75

GTC CTC TCT TTC CGT GGC TCT CGT TCC ATA GAA AAC TGG ATC GGA AAT 350 Val Leu Ser Phe Arg Gly Ser Arg Ser He Glu Asn Trp He Gly Asn 80 85 90

CTT AAC TTC GAC TTG AAA GAA ATA AAT GAC ATT TGC TCC GGC TGC AGG 398 Leu Asn Phe Asp Leu Lys Glu He Asn Asp He Cys Ser Gly Cys Arg 95 100 105

GGA CAT GAC GGC TTC ACC TCG AGC TGG AGG TCT GTA GCC GAT ACG TTA 446 Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu 110 115 120 125

AGG CAG AAG GTG GAG GAT GCT GTG AGG GAG CAT CCC GAC TAT CGC GTG 494 Arg Gin Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val 130 135 140

GTG TTT ACC GGA CAT AGC TTG GGT GGT GCA TTG GCA ACT GTT GCC GGA 542 Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly 145 150 155

GCA GAC CTG CGT GGA AAT GGG TAT GAC ATC GAC GTG TTT TCA TAT GGC 590 Ala Asp Leu Arg Gly Asn Gly Tyr Asp He Asp Val Phe Ser Tyr Gly 160 165 170

GCC CCC CGA GTC GGA AAC AGG GCT TTT GCA GAA TTC CTG ACC GTA CAG 638 Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gin 175 180 185

ACC GGC GGT ACC CTC TAC CGC ATT ACC CAC ACC AAT GAT ATT GTC CCT 686 Thr Gly Gly Thr Leu Tyr Arg He Thr His Thr Asn Asp He Val Pro 190 195 200 205 AGA CTC CCG CCG CGC GAG TTC GGT TAC AGC CAT TCT AGC CCA GAG TAC 734 Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr 210 215 220

TGG ATC AAA TCT GGA ACC CTT GTC CCC GTC ACC CGA AAC GAC ATC GTG 782 Trp He Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp He Val 225 230 235

AAG ATA GAA GGC ATC GAT GCC ACC GGC GGC AAT AAC CAG CCT AAC ATT 830 Lys He Glu Gly He Asp Ala Thr Gly Gly Asn Asn Gin Pro Asn He 240 245 250

CCG GAT ATC CCT GCG CAC CTA TGG TAC TTC GGG TTA ATT GGG ACA TGT 878 Pro Asp He Pro Ala His Leu Trp Tyr Phe Gly Leu He Gly Thr Cys 255 260 265

CTT TAGTGCGAAG CTT 894

Leu

270

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 270 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Ala Glu Val Ser Gin Asp Leu Phe Asn Gin Phe Asn Leu Phe Ala Gin

1 5 10 15

Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly 20 25 30

Thr Asn He Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala 35 40 45

Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val 50 55 60 Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu He Val Leu Ser 65 70 75 80

Phe Arg Gly Ser Arg Ser He Glu Asn Trp He Gly Asn Leu Asn Phe 85 90 95

Asp Leu Lys Glu He Asn Asp He Cys Ser Gly Cys Arg Gly His Asp 100 105 110

Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gin Lys 115 120 125

Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr 130 135 140

Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu 145 150 155 160

Arg Gly Asn Gly Tyr Asp He Asp Val Phe Ser Tyr Gly Ala Pro Arg 165 170 175

Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gin Thr Gly Gly 180 185 190

Thr Leu Tyr Arg He Thr His Thr Asn Asp He Val Pro Arg Leu Pro 195 200 205

Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp He Lys 210 215 220

Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp He Val Lys He Glu 225 230 235 240

Gly He Asp Ala Thr Gly Gly Asn Asn Gin Pro Asn He Pro Asp He 245 250 255

Pro Ala His Leu Trp Tyr Phe Gly Leu He Gly Thr Cys Leu 260 265 270 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE: (B) CLONE: primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

ATCCCTGCGC ACCTATGGTA CTTCGGGTTA ATTGGGACAT GTCTTGCTAG CCTTA 55

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE: (B) CLONE: primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

AGCTTAAGGC TAGCAAGACA TGTCCCAATT AACCCGAAGT ACCATAGGTG CGCAGGGAT 59

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1828 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: Geotrichum candidum

(B) STRAIN: CMICC 335426

(i ) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 40..1731

(D) OTHER INFORMATION: /product= "lipase"

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 40..96

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 97..1728

(D) OTHER INFORMATION: /product= "lipase" /gene= "lipB"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

AATTCGGCAC GAGATTCCTT TGATTTGCAA CTGTTAATC ATG GTT TCC AAA AGC 54

Met Val Ser Lys Ser -19 -15

TTT TTT TTG GCT GCG GCG CTC AAC GTA GTG GGC ACC TTG GCC CAG GCC 102 Phe Phe Leu Ala Ala Ala Leu Asn Val Val Gly Thr Leu Ala Gin Ala

-10 -5 1

CCC ACG GCC GTT CTT AAT GGC AAC GAG GTC ATC TCT GGT GTC CTT GAG 150 Pro Thr Ala Val Leu Asn Gly Asn Glu Val He Ser Gly Val Leu Glu 5 10 15

GGC AAG GTT GAT ACC TTC AAG GGA ATC CCA TTT GCT GAC CCT CCT GTT 198 Gly Lys Val Asp Thr Phe Lys Gly He Pro Phe Ala Asp Pro Pro Val 20 25 30

GGT GAC TTG CGG TTC AAG CAC CCC CAG CCT TTC ACT GGA TCC TAC CAG 246 Gly Asp Leu Arg Phe Lys His Pro Gin Pro Phe Thr Gly Ser Tyr Gin 35 40 45 50

GGT CTT AAG GCC AAC GAC TTC AGC TCT GCT TGT ATG CAG CTT GAT CCT 294 Gly Leu Lys Ala Asn Asp Phe Ser Ser Ala Cys Met Gin Leu Asp Pro 55 60 65 GGC AAT GCC TTT TCT TTG CTT GAC AAA GTA GTG GGC TTG GGA AAG ATT 342 Gly Asn Ala Phe Ser Leu Leu Asp Lys Val Val Gly Leu Gly Lys He 70 75 80

CTT CCT GAT AAC CTT AGA GGC CCT CTT TAT GAC ATG GCC CAG GGT AGT 390 Leu Pro Asp Asn Leu Arg Gly Pro Leu Tyr Asp Met Ala Gin Gly Ser 85 90 95

GTC TCC ATG AAT GAG GAC TGT CTC TAC CTT AAC GTT TTC CGC CCC GCT 438 Val Ser Met Asn Glu Asp Cys Leu Tyr Leu Asn Val Phe Arg Pro Ala 100 105 110

GGC ACC AAG CCT GAT GCT AAG CTC CCC GTC ATG GTT TGG ATT TAC GGT 486 Gly Thr Lys Pro Asp Ala Lys Leu Pro Val Met Val Trp He Tyr Gly 115 120 125 130

GGT GCC TTT GTG TTT GGT TCT TCT GCT TCT TAC CCT GGT AAC GGC TAC 534 Gly Ala Phe Val Phe Gly Ser Ser Ala Ser Tyr Pro Gly Asn Gly Tyr 135 140 145

GTC AAG GAG AGT GTG GAA ATG GGC CAG CCT GTT GTG TTT GTT TCC ATC 582 Val Lys Glu Ser Val Glu Met Gly Gin Pro Val Val Phe Val Ser He 150 155 160

AAC TAC CGT ACC GGC CCC TAT GGA TTC TTG GGT GGT GAT GCC ATC ACC 630 Asn Tyr Arg Thr Gly Pro Tyr Gly Phe Leu Gly Gly Asp Ala He Thr 165 170 175

GCT GAG GGC AAC ACC AAC GCT GGT CTG CAC GAC CAG CGC AAG GGT CTC 678 Ala Glu Gly Asn Thr Asn Ala Gly Leu His Asp Gin Arg Lys Gly Leu 180 185 190

GAG TGG GTT AGC GAC AAC ATT GCC AAC TTT GGT GGT GAT CCC GAC AAG 726 Glu Trp Val Ser Asp Asn He Ala Asn Phe Gly Gly Asp Pro Asp Lys 195 200 205 210

GTC ATG ATT TTC GGT GAG TCC GCT GGT GCC ATG AGT GTT GCT CAC CAG 774 Val Met He Phe Gly Glu Ser Ala Gly Ala Met Ser Val Ala His Gin 215 220 225

CTT GTT GCC TAC GGT GGT GAC AAC ACC TAC AAC GGA AAG CAG CTT TTC 822 Leu Val Ala Tyr Gly Gly Asp Asn Thr Tyr Asn Gly Lys Gin Leu Phe 230 235 240 CAC TCT GCC ATT CTT CAG TCT GGC GGT CCT CTT CCT TAC TTT GAC TCT 870 His Ser Ala He Leu Gin Ser Gly Gly Pro Leu Pro Tyr Phe Asp Ser 245 250 255

ACT TCT GTT GGT CCC GAG AGT GCC TAC AGC AGA TTT GCT CAG TAT GCC 918 Thr Ser Val Gly Pro Glu Ser Ala Tyr Ser Arg Phe Ala Gin Tyr Ala 260 265 270

GGA TGT GAC ACC AGT GCC AGT GAT AAT GAC ACT CTG GCT TGT CTC CGC 966 Gly Cys Asp Thr Ser Ala Ser Asp Asn Asp Thr Leu Ala Cys Leu Arg 275 280 285 290

AGC AAG TCC AGC GAT GTC TTG CAC AGT GCG CAG AAC TCG TAT GAT CTT 1014 Ser Lys Ser Ser Asp Val Leu His Ser Ala Gin Asn Ser Tyr Asp Leu 295 300 305

AAG GAC CTG TTT GGT CTG CTC CCT CAA TTC CTT GGA TTT GGT CCC AGA 1062 Lys Asp Leu Phe Gly Leu Leu Pro Gin Phe Leu Gly Phe Gly Pro Arg 310 315 320

CCC GAC GGC AAC ATT ATT CCC GAT GCC GCT TAT GAG CTC TAC CGC AGC 1110 Pro Asp Gly Asn He He Pro Asp Ala Ala Tyr Glu Leu Tyr Arg Ser 325 330 335

GGT AGA TAC GCC AAG GTT CCC TAC ATT ACT GGC AAC CAG GAG GAT GAG 1158 Gly Arg Tyr Ala Lys Val Pro Tyr He Thr Gly Asn Gin Glu Asp Glu 340 345 350

GGT ACT ATT CTT GCC CCC GTT GCT ATT AAT GCT ACC ACT ACT CCC CAT 1206 Gly Thr He Leu Ala Pro Val Ala He Asn Ala Thr Thr Thr Pro His 355 360 365 370

GTT AAG AAG TGG TTG AAG TAC ATT TGT AGC CAG GCT TCT GAC GCT TCG 1254 Val Lys Lys Trp Leu Lys Tyr He Cys Ser Gin Ala Ser Asp Ala Ser 375 380 385

CTT GAT CGT GTT TTG TCG CTC TAC CCC GGC TCT TGG TCG GAG GGT TCA 1302 Leu Asp Arg Val Leu Ser Leu Tyr Pro Gly Ser Trp Ser Glu Gly Ser 390 395 400

CCA TTC CGC ACT GGT ATT CTT AAT GCT CTT ACC CCT CAG TTC AAG CGC 1350 Pro Phe Arg Thr Gly He Leu Asn Ala Leu Thr Pro Gin Phe Lys Arg 405 410 415 ATT GCT GCC ATT TTC ACT GAT TTG CTG TTC CAG TCT CCT CGT CGT GTT 1398 He Ala Ala He Phe Thr Asp Leu Leu Phe Gin Ser Pro Arg Arg Val 420 425 430

ATG CTT AAC GCT ACC AAG GAC GTC AAC CGC TGG ACT TAC CTT GCC ACC 1446 Met Leu Asn Ala Thr Lys Asp Val Asn Arg Trp Thr Tyr Leu Ala Thr 435 440 445 450

CAG CTC CAT AAC CTC GTT CCA TTT TTG GGT ACT TTC CAT GGC AGT GAT 1494 Gin Leu His Asn Leu Val Pro Phe Leu Gly Thr Phe His Gly Ser Asp 455 460 465

CTT CTT TTT CAA TAC TAC GTG GAC CTT GGC CCA TCT TCT GCT TAC CGC 1542 Leu Leu Phe Gin Tyr Tyr Val Asp Leu Gly Pro Ser Ser Ala Tyr Arg 470 475 480

CGC TAC TTT ATC TCG TTT GCC AAC CAC CAC GAC CCC AAC GTT GGT ACC 1590 Arg Tyr Phe He Ser Phe Ala Asn His His Asp Pro Asn Val Gly Thr 485 490 495

AAC CTC CAA CAG TGG GAT ATG TAC ACT GAT GCA GGC AAG GAG ATG CTT 1638 Asn Leu Gin Gin Trp Asp Met Tyr Thr Asp Ala Gly Lys Glu Met Leu 500 505 510

CAG ATT CAT ATG ATT GGT AAC TCT ATG AGA ACT GAC GAC TTT AGA ATC 1686 Gin He His Met He Gly Asn Ser Met Arg Thr Asp Asp Phe Arg He 515 520 525 530

GAG GGA ATC TCG AAC TTT GAG TCT GAC GTT ACT CTC TTC GGT TAATCCCATT 1738 Glu Gly He Ser Asn Phe Glu Ser Asp Val Thr Leu Phe Gly

535 540 545

TAGCAAGTTT TGTGTATTTC AAGTATACCA GTTGATGTAA TATATCAATA GATTACAAAT 1798

TAATTAGTGA AAAAAAAAAA AAAAAAAAAC ' 1828

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 563 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Val Ser Lys Ser Phe Phe Leu Ala Ala Ala Leu Asn Val Val Gly -19 -15 -10 -5

Thr Leu Ala Gin Ala Pro Thr Ala Val Leu Asn Gly Asn Glu Val He 1 5 10

Ser Gly Val Leu Glu Gly Lys Val Asp Thr Phe Lys Gly He Pro Phe 15 20 25

Ala Asp Pro Pro Val Gly Asp Leu Arg Phe Lys His Pro Gin Pro Phe 30 35 40 45

Thr Gly Ser Tyr Gin Gly Leu Lys Ala Asn Asp Phe Ser Ser Ala Cys 50 55 60

Met Gin Leu Asp Pro Gly Asn Ala Phe Ser Leu Leu Asp Lys Val Val 65 70 75

Gly Leu Gly Lys He Leu Pro Asp Asn Leu Arg Gly Pro Leu Tyr Asp 80 85 90

Met Ala Gin Gly Ser Val Ser Met Asn Glu Asp Cys Leu Tyr Leu Asn 95 100 105

Val Phe Arg Pro Ala Gly Thr Lys Pro Asp Ala Lys Leu Pro Val Met 110 115 120 125

Val Trp He Tyr Gly Gly Ala Phe Val Phe Gly Ser Ser Ala Ser Tyr 130 135 140

Pro Gly Asn Gly Tyr Val Lys Glu Ser Val Glu Met Gly Gin Pro Val 145 150 155

Val Phe Val Ser He Asn Tyr Arg Thr Gly Pro Tyr Gly Phe Leu Gly 160 165 170

Gly Asp Ala He Thr Ala Glu Gly Asn Thr Asn Ala Gly Leu His Asp 175 180 185

Gin Arg Lys Gly Leu Glu Trp Val Ser Asp Asn He Ala Asn Phe Gly 190 195 200 205 Gly Asp Pro Asp Lys Val Met He Phe Gly Glu Ser Ala Gly Ala Met 210 215 220

Ser Val Ala His Gin Leu Val Ala Tyr Gly Gly Asp Asn Thr Tyr Asn 225 230 235

Gly Lys Gin Leu Phe His Ser Ala He Leu Gin Ser Gly Gly Pro Leu 240 245 250

Pro Tyr Phe Asp Ser Thr Ser Val Gly Pro Glu Ser Ala Tyr Ser Arg 255 260 265

Phe Ala Gin Tyr Ala Gly Cys Asp Thr Ser Ala Ser Asp Asn Asp Thr 270 275 280 285

Leu Ala Cys Leu Arg Ser Lys Ser Ser Asp Val Leu His Ser Ala Gin 290 295 300

Asn Ser Tyr Asp Leu Lys Asp Leu Phe Gly Leu Leu Pro Gin Phe Leu 305 310 315

Gly Phe Gly Pro Arg Pro Asp Gly Asn He He Pro Asp Ala Ala Tyr 320 325 330

Glu Leu Tyr Arg Ser Gly Arg Tyr Ala Lys Val Pro Tyr He Thr Gly 335 340 345

Asn Gin Glu Asp Glu Gly Thr He Leu Ala Pro Val Ala He Asn Ala 350 355 360 365

Thr Thr Thr Pro His Val Lys Lys Trp Leu Lys Tyr He Cys Ser Gin 370 375 380

Ala Ser Asp Ala Ser Leu Asp Arg Val Leu Ser Leu Tyr Pro Gly Ser 385 390 395

Trp Ser Glu Gly Ser Pro Phe Arg Thr Gly He Leu Asn Ala Leu Thr 400 405 410

Pro Gin Phe Lys Arg He Ala Ala He Phe Thr Asp Leu Leu Phe Gin 415 420 425

Ser Pro Arg Arg Val Met Leu Asn Ala Thr Lys Asp Val Asn Arg Trp 430 435 440 445 Thr Tyr Leu Ala Thr Gin Leu His Asn Leu Val Pro Phe Leu Gly Thr 450 455 460

Phe His Gly Ser Asp Leu Leu Phe Gin Tyr Tyr Val Asp Leu Gly Pro 465 470 475

Ser Ser Ala Tyr Arg Arg Tyr Phe He Ser Phe Ala Asn His His Asp 480 485 490

Pro Asn Val Gly Thr Asn Leu Gin Gin Trp Asp Met Tyr Thr Asp Ala 495 500 505

Gly Lys Glu Met Leu Gin He His Met He Gly Asn Ser Met Arg Thr 510 515 520 525

Asp Asp Phe Arg He Glu Gly He Ser Asn Phe Glu Ser Asp Val Thr 530 535 540

Leu Phe Gly

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: primer lipo3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

GGGGCGGCCG CGCAGGCCCC AAGGCGGTCT CTCAAT 36 (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Part non-coding strand lipasell

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

ATTGAGAGAC CGCCGTGGGG CCTGGGCCAG 30

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Part coding strand lipasell

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

CAAACTTTGA GACTGACGTT AATCTCTACG GTTAAAAC 38

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:

(B) CLONE: primer lipo4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CCCCGCTAGC ACCGTAGAGA TTAACGTCAG TC 32

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: primer lipoδ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

CCCGCGGCCG CGAGCATTGA TGGTGGTATC 30

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Part non-coding strand lipase

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GATACCACGA TCAATGCT 18 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Part coding strand lipase

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

AACACAGGCC TCTGTACT 18

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: primer lipoδ

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

CCGCGCTAGC AGTACAGAGG CCTGTGTT 28

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2685 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Saccharomyces cerevisiae

(vii) IMMEDIATE SOURCE:

(B) CLONE: pYY105

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..2685

(D) OTHER INFORMATION: /product= "Flocculation protein" /gene= "FLOl"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

ATG ACA ATG CCT CAT CGC TAT ATG TTT TTG GCA GTC TTT ACA CTT CTG 48 Met Thr Met Pro His Arg Tyr Met Phe Leu Ala Val Phe Thr Leu Leu 1 5 10 15

GCA CTA ACT AGT GTG GCC TCA GGA GCC ACA GAG GCG TGC TTA CCA GCA 96 Ala Leu Thr Ser Val Ala Ser Gly Ala Thr Glu Ala Cys Leu Pro Ala 20 25 30

GGC CAG AGG AAA AGT GGG ATG AAT ATA AAT TTT TAC CAG TAT TCA TTG 144 Gly Gin Arg Lys Ser Gly Met Asn He Asn Phe Tyr Gin Tyr Ser Leu

35 40 45

AAA GAT TCC TCC ACA TAT TCG AAT GCA GCA TAT ATG GCT TAT GGA TAT 192 Lys Asp Ser Ser Thr Tyr Ser Asn Ala Ala Tyr Met Ala Tyr Gly Tyr 50 55 60

GCC TCA AAA ACC AAA CTA GGT TCT GTC GGA GGA CAA ACT GAT ATC TCG 240 Ala Ser Lys Thr Lys Leu Gly Ser Val Gly Gly Gin Thr Asp He Ser 65 70 75 80

ATT GAT TAT AAT ATT CCC TGT GTT AGT TCA TCA GGC ACA TTT CCT TGT 288 He Asp Tyr Asn He Pro Cys Val Ser Ser Ser Gly Thr Phe Pro Cys 85 90 95

CCT CAA GAA GAT TCC TAT GGA AAC TGG GGA TGC AAA GGA ATG GGT GCT 336 Pro Gin Glu Asp Ser Tyr Gly Asn Trp Gly Cys Lys Gly Met Gly Ala 100 105 110

TGT TCT AAT AGT CAA GGA ATT GCA TAC TGG AGT ACT GAT TTA TTT GGT 384 Cys Ser Asn Ser Gin Gly He Ala Tyr Trp Ser Thr Asp Leu Phe Gly 115 120 125 TTC TAT ACT ACC CCA ACA AAC GTA ACC CTA GAA ATG ACA GGT TAT TTT 432 Phe Tyr Thr Thr Pro Thr Asn Val Thr Leu Glu Met Thr Gly Tyr Phe 130 135 140

TTA CCA CCA CAG ACG GGT TCT TAC ACA TTC AAG TTT GCT ACA GTT GAC 480 Leu Pro Pro Gin Thr Gly Ser Tyr Thr Phe Lys Phe Ala Thr Val Asp 145 150 155 160

GAC TCT GCA ATT CTA TCA GTA GGT GGT GCA ACC GCG TTC AAC TGT TGT 528 Asp Ser Ala He Leu Ser Val Gly Gly Ala Thr Ala Phe Asn Cys Cys 165 170 175

GCT CAA CAG CAA CCG CCG ATC ACA TCA ACG AAC TTT ACC ATT GAC GGT 576 Ala Gin Gin Gin Pro Pro He Thr Ser Thr Asn Phe Thr He Asp Gly 180 185 190

ATC AAG CCA TGG GGT GGA AGT TTG CCA CCT AAT ATC GAA GGA ACC GTC 624 He Lys Pro Trp Gly Gly Ser Leu Pro Pro Asn He Glu Gly Thr Val 195 200 205

TAT ATG TAC GCT GGC TAC TAT TAT CCA ATG AAG GTT GTT TAC TCG AAC 672 Tyr Met Tyr Ala Gly Tyr Tyr Tyr Pro Met Lys Val Val Tyr Ser Asn 210 215 220

GCT GTT TCT TGG GGT ACA CTT CCA ATT AGT GTG ACA CTT CCA GAT GGT 720 Ala Val Ser Trp Gly Thr Leu Pro He Ser Val Thr Leu Pro Asp Gly 225 230 235 240

ACC ACT GTA AGT GAT GAC TTC GAA GGG TAC GTC TAT TCC TTT GAC GAT 768 Thr Thr Val Ser Asp Asp Phe Glu Gly Tyr Val Tyr Ser Phe Asp Asp 245 250 255

GAC CTA AGT CAA TCT AAC TGT ACT GTC CCT GAC CCT TCA AAT TAT GCT 816 Asp Leu Ser Gin Ser Asn Cys Thr Val Pro Asp Pro Ser Asn Tyr Ala 260 265 270

GTC AGT ACC ACT ACA ACT ACA ACG GAA CCA TGG ACC GGT ACT TTC ACT 864 Val Ser Thr Thr Thr Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr 275 280 285

TCT ACA TCT ACT GAA ATG ACC ACC GTC ACC GGT ACC AAC GGC GTT CCA 912 Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Val Pro 290 295 300 ACT GAC GAA ACC GTC ATT GTC ATC AGA ACT CCA ACC AGT GAA GGT CTA 960 Thr Asp Glu Thr Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu 305 310 315 320

ATC AGC ACC ACC ACT GAA CCA TGG ACT GGC ACT TTC ACT TCG ACT TCC 1008 He Ser Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser 325 330 335

ACT GAG GTT ACC ACC ATC ACT GGA ACC AAC GGT CAA CCA ACT GAC GAA 1056 Thr Glu Val Thr Thr He Thr Gly Thr Asn Gly Gin Pro Thr Asp Glu 340 345 350

ACT GTG ATT GTT ATC AGA ACT CCA ACC AGT GAA GGT CTA ATC AGC ACC 1104 Thr Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu He Ser Thr 355 360 365

ACC ACT GAA CCA TGG ACT GGT ACT TTC ACT TCT ACA TCT ACT GAA ATG 1152 Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr Glu Met 370 375 380

ACC ACC GTC ACC GGT ACT AAC GGT CAA CCA ACT GAC GAA ACC GTG ATT 1200 Thr Thr Val Thr Gly Thr Asn Gly Gin Pro Thr Asp Glu Thr Val He 385 390 395 400

GTT ATC AGA ACT CCA ACC AGT GAA GGT TTG GTT ACA ACC ACC ACT GAA 1248 Val He Arg Thr Pro Thr Ser Glu Gly Leu Val Thr Thr Thr Thr Glu 405 410 415

CCA TGG ACT GGT ACT TTT ACT TCG ACT TCC ACT GAA ATG TCT ACT GTC 1296 Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr Glu Met Ser Thr Val 420 425 430

ACT GGA ACC AAT GGC TTG CCA ACT GAT GAA ACT GTC ATT GTT GTC AAA 1344 Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Thr Val He Val Val Lys 435 440 445

ACT CCA ACT ACT GCC ATC TCA TCC AGT TTG TCA TCA TCA TCT TCA GGA 1392 Thr Pro Thr Thr Ala He Ser Ser Ser Leu Ser Ser Ser Ser Ser Gly 450 455 460

CAA ATC ACC AGC TCT ATC ACG TCT TCG CGT CCA ATT ATT ACC CCA TTC 1440 Gin He Thr Ser Ser He Thr Ser Ser Arg Pro He He Thr Pro Phe 465 470 475 480 TAT CCT AGC AAT GGA ACT TCT GTG ATT TCT TCC TCA GTA ATT TCT TCC 1488 Tyr Pro Ser Asn Gly Thr Ser Val He Ser Ser Ser Val He Ser Ser 485 490 495

TCA GTC ACT TCT TCT CTA TTC ACT TCT TCT CCA GTC ATT TCT TCC TCA 1536 Ser Val Thr Ser Ser Leu Phe Thr Ser Ser Pro Val He Ser Ser Ser 500 505 510

GTC ATT TCT TCT TCT ACA ACA ACC TCC ACT TCT ATA TTT TCT GAA TCA 1584 Val He Ser Ser Ser Thr Thr Thr Ser Thr Ser He Phe Ser Glu Ser 515 520 525

TCT AAA TCA TCC GTC ATT CCA ACC AGT AGT TCC ACC TCT GGT TCT TCT 1632 Ser Lys Ser Ser Val He Pro Thr Ser Ser Ser Thr Ser Gly Ser Ser 530 535 540

GAG AGC GAA ACG AGT TCA GCT GGT TCT GTC TCT TCT TCC TCT TTT ATC 1680 Glu Ser Glu Thr Ser Ser Ala Gly Ser Val Ser Ser Ser Ser Phe He 545 550 555 560

TCT TCT GAA TCA TCA AAA TCT CCT ACA TAT TCT TCT TCA TCA TTA CCA 1728 Ser Ser Glu Ser Ser Lys Ser Pro Thr Tyr Ser Ser Ser Ser Leu Pro 565 570 575

CTT GTT ACC AGT GCG ACA ACA AGC CAG GAA ACT GCT TCT TCA TTA CCA 1776 Leu Val Thr Ser Ala Thr Thr Ser Gin Glu Thr Ala Ser Ser Leu Pro 580 585 590

CCT GCT ACC ACT ACA AAA ACG AGC GAA CAA ACC ACT TTG GTT ACC GTG 1824 Pro Ala Thr Thr Thr Lys Thr Ser Glu Gin Thr Thr Leu Val Thr Val 595 600 605

ACA TCC TGC GAG TCT CAT GTG TGC ACT GAA TCC ATC TCC CCT GCG ATT 1872 Thr Ser Cys Glu Ser His Val Cys Thr Glu Ser He Ser Pro Ala He 610 615 620

GTT TCC ACA GCT ACT GTT ACT GTT AGC GGC GTC ACA ACA GAG TAT ACC 1920

Val Ser Thr Ala Thr Val Thr Val Ser Gly Val Thr Thr Glu Tyr Thr 625 630 635 640

ACA TGG TGC CCT ATT TCT ACT ACA GAG ACA ACA AAG CAA ACC AAA GGG 1968 Thr Trp Cys Pro He Ser Thr Thr Glu Thr Thr Lys Gin Thr Lys Gly 645 650 655 ACA ACA GAG CAA ACC ACA GAA ACA ACA AAA CAA ACC ACG GTA GTT ACA 2016 Thr Thr Glu Gin Thr Thr Glu Thr Thr Lys Gin Thr Thr Val Val Thr 660 665 670

ATT TCT TCT TGT GAA TCT GAC GTA TGC TCT AAG ACT GCT TCT CCA GCC 2064 He Ser Ser Cys Glu Ser Asp Val Cys Ser Lys Thr Ala Ser Pro Ala 675 680 685

ATT GTA TCT ACA AGC ACT GCT ACT ATT AAC GGC GTT ACT ACA GAA TAC 2112 He Val Ser Thr Ser Thr Ala Thr He Asn Gly Val Thr Thr Glu Tyr 690 695 700

ACA ACA TGG TGT CCT ATT TCC ACC ACA GAA TCG AGG CAA CAA ACA ACG 2160 Thr Thr Trp Cys Pro He Ser Thr Thr Glu Ser Arg Gin Gin Thr Thr 705 710 715 720

CTA GTT ACT GTT ACT TCC TGC GAA TCT GGT GTG TGT TCC GAA ACT GCT 2208 Leu Val Thr Val Thr Ser Cys Glu Ser Gly Val Cys Ser Glu Thr Ala 725 730 735

TCA CCT GCC ATT GTT TCG ACG GCC ACG GCT ACT GTG AAT GAT GTT GTT 2256 Ser Pro Ala He Val Ser Thr Ala Thr Ala Thr Val Asn Asp Val Val 740 745 750

ACG GTC TAT CCT ACA TGG AGG CCA CAG ACT GCG AAT GAA GAG TCT GTC 2304 Thr Val Tyr Pro Thr Trp Arg Pro Gin Thr Ala Asn Glu Glu Ser Val 755 760 765

AGC TCT AAA ATG AAC AGT GCT ACC GGT GAG ACA ACA ACC AAT ACT TTA 2352 Ser Ser Lys Met Asn Ser Ala Thr Gly Glu Thr Thr Thr Asn Thr Leu 770 775 780

GCT GCT GAA ACG ACT ACC AAT ACT GTA GCT GCT GAG ACG ATT ACC AAT 2400 Ala Ala Glu Thr Thr Thr Asn Thr Val Ala Ala Glu Thr He Thr Asn 785 790 795 800

ACT GGA GCT GCT GAG ACG AAA ACA GTA GTC ACC TCT TCG CTT TCA AGA 2448 Thr Gly Ala Ala Glu Thr Lys Thr Val Val Thr Ser Ser Leu Ser Arg 805 810 815

TCT AAT CAC GCT GAA ACA CAG ACG GCT TCC GCG ACC GAT GTG ATT GGT 2496 Ser Asn His Ala Glu Thr Gin Thr Ala Ser Ala Thr Asp Val He Gly 820 825 830 CAC AGC AGT AGT GTT GTT TCT GTA TCC GAA ACT GGC AAC ACC AAG AGT 2544 His Ser Ser Ser Val Val Ser Val Ser Glu Thr Gly Asn Thr Lys Ser 835 840 845

CTA ACA AGT TCC GGG TTG AGT ACT ATG TCG CAA CAG CCT CGT AGC ACA 2592 Leu Thr Ser Ser Gly Leu Ser Thr Met Ser Gin Gin Pro Arg Ser Thr 850 855 860

CCA GCA AGC AGC ATG GTA GGA TAT AGT ACA GCT TCT TTA GAA ATT TCA 2640 Pro Ala Ser Ser Met Val Gly Tyr Ser Thr Ala Ser Leu Glu He Ser 865 870 875 880

ACG TAT GCT GGC AGT GCA ACA GCT TAC TGG CCG GTA GTG GTT TAA 2686

Thr Tyr Ala Gly Ser Ala Thr Ala Tyr Trp Pro Val Val Val

885 890 895

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 894 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Met Thr Met Pro His Arg Tyr Met Phe Leu Ala Val Phe Thr Leu Leu 1 5 10 15

Ala Leu Thr Ser Val Ala Ser Gly Ala Thr Glu Ala Cys Leu Pro Ala 20 25 30

Gly Gin Arg Lys Ser Gly Met Asn He Asn Phe Tyr Gin Tyr Ser Leu 35 40 45

Lys Asp Ser Ser Thr Tyr Ser Asn Ala Ala Tyr Met Ala Tyr Gly Tyr 50 55 60

Ala Ser Lys Thr Lys Leu Gly Ser Val Gly Gly Gin Thr Asp He Ser 65 70 75 80

He Asp Tyr Asn He Pro Cys Val Ser Ser Ser Gly Thr Phe Pro Cys 85 90 95 Pro Gin Glu Asp Ser Tyr Gly Asn Trp Gly Cys Lys Gly Met Gly Ala 100 105 110

Cys Ser Asn Ser Gin Gly He Ala Tyr Trp Ser Thr Asp Leu Phe Gly 115 120 125

Phe Tyr Thr Thr Pro Thr Asn Val Thr Leu Glu Met Thr Gly Tyr Phe 130 135 140

Leu Pro Pro Gin Thr Gly Ser Tyr Thr Phe Lys Phe Ala Thr Val Asp 145 150 155 160

Asp Ser Ala He Leu Ser Val Gly Gly Ala Thr Ala Phe Asn Cys Cys 165 170 175

Ala Gin Gin Gin Pro Pro He Thr Ser Thr Asn Phe Thr He Asp Gly 180 185 190

He Lys Pro Trp Gly Gly Ser Leu Pro Pro Asn He Glu Gly Thr Val 195 200 205

Tyr Met Tyr Ala Gly Tyr Tyr Tyr Pro Met Lys Val Val Tyr Ser Asn 210 215 220

Ala Val Ser Trp Gly Thr Leu Pro He Ser Val Thr Leu Pro Asp Gly 225 230 235 240

Thr Thr Val Ser Asp Asp Phe Glu Gly Tyr Val Tyr Ser Phe Asp Asp 245 250 255

Asp Leu Ser Gin Ser Asn Cys Thr Val Pro Asp Pro Ser Asn Tyr Ala 260 265 270

Val Ser Thr Thr Thr Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr 275 280 285

Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gly Thr Asn Gly Val Pro 290 295 300

Thr Asp Glu Thr Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu 305 310 315 320

He Ser Thr Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser 325 330 335 Thr Glu Val Thr Thr He Thr Gly Thr Asn Gly Gin Pro Thr Asp Glu 340 345 350

Thr Val He Val He Arg Thr Pro Thr Ser Glu Gly Leu He Ser Thr 355 360 365

Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr Glu Met 370 375 380

Thr Thr Val Thr Gly Thr Asn Gly Gin Pro Thr Asp Glu Thr Val He 385 390 395 400

Val He Arg Thr Pro Thr Ser Glu Gly Leu Val Thr Thr Thr Thr Glu 405 410 415

Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Thr Glu Met Ser Thr Val 420 425 430

Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Thr Val He Val Val Lys 435 440 445

Thr Pro Thr Thr Ala He Ser Ser Ser Leu Ser Ser Ser Ser Ser Gly 450 455 460

Gin He Thr Ser Ser He Thr Ser Ser Arg Pro He He Thr Pro Phe 465 470 475 480

Tyr Pro Ser Asn Gly Thr Ser Val He Ser Ser Ser Val He Ser Ser 485 490 495

Ser Val Thr Ser Ser Leu Phe Thr Ser Ser Pro Val He Ser Ser Ser 500 505 510

Val He Ser Ser Ser Thr Thr Thr Ser Thr Ser He Phe Ser Glu Ser 515 520 525

Ser Lys Ser Ser Val He Pro Thr Ser Ser Ser Thr Ser Gly Ser Ser 530 535 540

Glu Ser Glu Thr Ser Ser Ala Gly Ser Val Ser Ser Ser Ser Phe He 545 550 555 560

Ser Ser Glu Ser Ser Lys Ser Pro Thr Tyr Ser Ser Ser Ser Leu Pro 565 570 575 Leu Val Thr Ser Ala Thr Thr Ser Gin Glu Thr Ala Ser Ser Leu Pro 580 585 590

Pro Ala Thr Thr Thr Lys Thr Ser Glu Gin Thr Thr Leu Val Thr Val 595 600 605

Thr Ser Cys Glu Ser His Val Cys Thr Glu Ser He Ser Pro Ala He 610 615 620

Val Ser Thr Ala Thr Val Thr Val Ser Gly Val Thr Thr Glu Tyr Thr 625 630 635 640

Thr Trp Cys Pro He Ser Thr Thr Glu Thr Thr Lys Gin Thr Lys Gly 645 650 655

Thr Thr Glu Gin Thr Thr Glu Thr Thr Lys Gin Thr Thr Val Val Thr 660 665 670

He Ser Ser Cys Glu Ser Asp Val Cys Ser Lys Thr Ala Ser Pro Ala 675 680 685

He Val Ser Thr Ser Thr Ala Thr He Asn Gly Val Thr Thr Glu Tyr 690 695 700

Thr Thr Trp Cys Pro He Ser Thr Thr Glu Ser Arg Gin Gin Thr Thr 705 710 715 720

Leu Val Thr Val Thr Ser Cys Glu Ser Gly Val Cys Ser Glu Thr Ala 725 730 735

Ser Pro Ala He Val Ser Thr Ala Thr Ala Thr Val Asn Asp Val Val 740 745 750

Thr Val Tyr Pro Thr Trp Arg Pro Gin Thr Ala Asn Glu Glu Ser Val 755 760 765

Ser Ser Lys Met Asn Ser Ala Thr Gly Glu Thr Thr Thr Asn Thr Leu 770 775 780

Ala Ala Glu Thr Thr Thr Asn Thr Val Ala Ala Glu Thr He Thr Asn 785 790 795 800

Thr Gly Ala Ala Glu Thr Lys Thr Val Val Thr Ser Ser Leu Ser Arg 805 810 815 Ser Asn His Ala Glu Thr Gin Thr Ala Ser Ala Thr Asp Val He Gly 820 825 830

His Ser Ser Ser Val Val Ser Val Ser Glu Thr Gly Asn Thr Lys Ser 835 840 845

Leu Thr Ser Ser Gly Leu Ser Thr Met Ser Gin Gin Pro Arg Ser Thr 850 855 860

Pro Ala Ser Ser Met Val Gly Tyr Ser Thr Ala Ser Leu Glu He Ser 865 870 875 880

Thr Tyr Ala Gly Ser Ala Thr Ala Tyr Trp Pro Val Val Val 885 890

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: primer pcrflol

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

GAATTCGCTA GCAATTATGC TGTCAGTACC 30

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:

(B) CLONE: Part non-coding sequence FLOl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

AGTGGTACTG ACAGCATAAT TTGA 24

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Part coding sequence FLOl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

AATAAAATTC GCGTTCTTTT TACG 24

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: primer pcrflo2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

GAGCTCAAGC TTCGTAAAAA GAACGCGAAT T 31

Claims

1. A method for immobilizing an enzyme, comprising the use of recombinant DNA techniques for producing an enzyme or a functional part thereof linked to the ce wall of a host cell, preferably a microbial cell, and whereby the enzyme or functional fragment thereof is localized at the exterior of the cell wall.

2. The method of claim 1, wherein the enzyme or the functional part thereof is immobilized by linking to the C-terminal part of a protein that ensures anchorin in the cell wall.

3. A recombinant polynucleotide comprising a structural gene encoding a protein providing catalytic activity and at least a part of a gene encoding a protein capable of anchoring in a eukaryotic or prokaryotic cell wall, said part encoding at least the C-terminal part of said anchoring protein.

4. The polynucleotide of claim 3, further comprising a sequence encoding a signal peptide ensuring secretion of the expression product of the polynucleotide.

5. The polynucleotide of claim 4, wherein the signal peptide is derived from a protein selected from the group consisting of glycosyl-phosphatidyl-inositol (GPI) anchoring protein, α-factor, α-agglutinin, invertase or inulinase, α-amylase of Bacillus, and proteinases of lactic acid bacteria.

6. The polynucleotide of any of claims 3-5, wherein the protein capable of anchori in the cell wall is selected from the group consisting of α-agglutinin, AGA1, FLOl, Major Cell Wall Protein of lower eukaryotes, and proteinases of lactic acid bacteria.

7. The polynucleotide of any of claims 3-6, operably linked to a promoter, preferably an inducible promoter.

8. The polynucleotide of any of claims 3-7, wherein the protein providing catalytic activity is a hydrolytic enzyme, e.g. a lipase.

9. The polynucleotide of any of claims 3-7, wherein the protein providing catalytic activity is an oxidoreductase, e.g. an oxidase.

10. A recombinant vector comprising a polynucleotide as claimed in any of claims 3-9.

11. The recombinant vector of claim 10, wherein the protein providing catalytic activity exhibits said catalytic activity when present in a multimeric form, said vector further comprising a second polynucleotide comprising a structural gene encoding the same protein providing catalytic activity combined with a sequence encoding a signal peptide ensuring secretion of the expression product of said second polynucleotide, said second polynucleotide being operably linked to a regulatable promoter, preferably an inducible or repressible promoter.

12. A chimeric protein encoded by a polynucleotide as claimed in any of claims 3-9.

13. A host cell, preferably a microorganism, containing a polynucleotide as claimed i any of claims 3-9 or a vector as claimed in claim 10 or 11.

14. A host cell, preferably a microorganism, containing a polynucleotide as claimed i any of claims 3-9 or a vector as claimed in claim 10, wherein the protein providing catalytic activity exhibits said catalytic activity when present in a multimeric form, said microorganism further comprising a second polynucleotide comprising a structural gene encoding the same protein providing catalytic activit combined with a sequence encoding a signal peptide ensuring secretion of the expression product of said second polynucleotide, said second polynucleotide being operably linked to a regulatable promoter, preferably an inducible or repressible promoter and said second polynucleotide being present either in another vector or in the chromosome of said microorganism.

15. The host cell or microorganism of claim 13 or 14, having at least one of said polynucleotides integrated in its chromosome.

16. A host cell, preferably a microorganism, having a protein as claimed in claim 12 immobilized on its cell wall.

17. The host cell or microorganism of any of claims 13-16, which is a lower eukaryote, in particular a yeast.

18. A process for carrying out an enzymatic process by using an immobilized catalytically active protein, wherein a substrate for said catalytically active protein is contacted with a host cell or microorganism as claimed in any of claims 13-17.

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